DNA aptamer against EV-A71 VP1 protein: selection and application

Abstract Background Enterovirus 71 (EV-A71) is a highly infectious pathogen associated with hand, foot and mouth disease, herpangina, and various neurological complications, so it is important for the early detection and treatment of EV-A71. An aptamer is a nucleotide sequence that screened in vitro by the technology named systematic evolution of ligands by exponential enrichment technology (SELEX). Similar to antibodies, aptamers can bind to the targets with high specificity and affinity. Besides, emerging aptamers have many advantages comparing with antibodies, such as ease of synthesis and modification, having a wide variety of target materials, low manufacturing cost and easy flexibility in amending. Therefore, aptamers are promising in virus detection and anti-virus therapy. Methods Aptamers were selected by SELEX. Specificity, affinity and second structure were used to characterize the selected aptamers. Chemiluminescence was adopted to build an aptamer-based detection method for EV-A71. Cytopathogenic effects trial, the level of intracellular EV-A71 RNA and protein expression were used to evaluate the antiviral effect of the selected aptamers. Results Three DNA aptamers with high specificity and affinity for EV-A71structual protein VP1 were screened out. A rapid chemiluminutesescence aptamer biosensor for EV-A71 detection was designed out. The selected aptamers could inhibit the RNA replication and protein expression of EV-A71 in RD cells and ameliorate the cytopathogenic effects. Conclusions The aptamers against EV-A71 have the potentiality to be applied as attractive candidates used for EV-A71 detection and treatment in the future.

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Antidepressant Sertraline is a Broad-Spectrum Inhibitor of Enteroviruses Targeting Viral Entry Through Neutralization of Endolysosomal Acidification

10.20944/preprints202112.0203.v1 ◽

2021 ◽

Author(s):

Kuan-Chi Tseng ◽

Bang-Yan Hsu ◽

Pin Ling ◽

Wen-Wen Lu ◽

Cheng-Wen Lin ◽

...

Keyword(s):

Antiviral Activity ◽

Viral Entry ◽

Broad Spectrum ◽

Culture Media ◽

Antiviral Drug ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Antiviral Effect ◽

Neurological Complications ◽

Low Ph

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 to treat EV71 infections. In this study, we conducted an 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 relieved greatly by exposing virus-infected cells to extracellular low-pH culture media. Together, we have identified an FDA-approved antidepressant with the new indication for the broad-spectrum EV inhibition by blocking viral entry through the alkalization of the endolysosomal route.

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Antiviral Efficacy of Flavonoids against Enterovirus 71 Infection in Vitro and in Newborn Mice

Viruses ◽

10.3390/v11070625 ◽

2019 ◽

Vol 11 (7) ◽

pp. 625 ◽

Cited By ~ 16

Author(s):

Wenwen Dai ◽

Jinpeng Bi ◽

Fang Li ◽

Shuai Wang ◽

Xinyu Huang ◽

...

Keyword(s):

Protective Efficacy ◽

Lethal Dose ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Neurological Complications ◽

Ev71 Infection ◽

Newborn Mice ◽

Reported Data

Enterovirus 71 (EV71) infection is known to cause hand, foot, and mouth disease (HFMD), which is associated with neurological complications; however, there is currently no effective treatment for this infection. Flavonoids are a large group of naturally occurring compounds with multiple bioactivities, and the inhibitory effects of several flavonoids against EV71 have been studied in cell cultures; however, to date, there are no reported data on their effects in animal models. In this study, we confirmed the in vitro activities of eight flavonoids against EV71 infection, based on the inhibition of cytopathic effects. Moreover, these flavonoids were found to reduce viral genomic RNA replication and protein synthesis. We further demonstrated the protective efficacy of these flavonoids in newborn mice challenged with a lethal dose of EV71. Apigenin, luteolin, kaempferol, formononetin, and penduletin conferred survival protection of 88.89%, 91.67%, 88.89%, 75%, and 66.67%, respectively, from the lethal EV71 challenge. In addition, isorhamnetin provided the highest mice survival protection of 100% at a dose of 10 mg/kg. This study, to the best of our knowledge, is the first to evaluate the in vivo anti-EV7l activities of multiple flavonoids, and we accordingly identified flavonoids as potential leading compounds for anti-EV71 drug development.

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Antidepressant Sertraline Is a Broad-Spectrum Inhibitor of Enteroviruses Targeting Viral Entry through Neutralization of Endolysosomal Acidification

Viruses ◽

10.3390/v14010109 ◽

2022 ◽

Vol 14 (1) ◽

pp. 109

Author(s):

Kuan-Chi Tseng ◽

Bang-Yan Hsu ◽

Pin Ling ◽

Wen-Wen Lu ◽

Cheng-Wen Lin ◽

...

Keyword(s):

Antiviral Activity ◽

Viral Entry ◽

Broad Spectrum ◽

Culture Media ◽

Antiviral Drug ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Antiviral Effect ◽

Neurological Complications ◽

Low Ph

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.

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Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽

10.3390/v13020308 ◽

2021 ◽

Vol 13 (2) ◽

pp. 308

Author(s):

Ying-Ray Lee ◽

Chia-Ming Chang ◽

Yuan-Chieh Yeh ◽

Chi-Ying F. Huang ◽

Feng-Mao Lin ◽

...

Keyword(s):

Protein Expression ◽

Western Blotting ◽

Expression Profiles ◽

Plaque Assay ◽

Virus Titer ◽

Enterovirus 71 ◽

Luciferase Reporter ◽

Pathogen Invasion

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.

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In VitroAssessment of Combinations of Enterovirus Inhibitors against Enterovirus 71

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01073-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5357-5367 ◽

Cited By ~ 9

Author(s):

Yizhuo Wang ◽

Guiming Li ◽

Shilin Yuan ◽

Qianqian Gao ◽

Ke Lan ◽

...

Keyword(s):

Infectious Clone ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Antiviral Effect ◽

Mechanisms Of Action ◽

Rational Basis ◽

Inhibitory Mechanism ◽

Antiviral Therapies ◽

Resistant Phenotype ◽

Combination Regimens

ABSTRACTEnterovirus 71 (EV-A71) is a major causative pathogen of hand, foot, and mouth disease (HFMD) epidemics. No antiviral therapies are currently available for treating EV-A71 infections. Here, we selected five reported enterovirus inhibitors (suramin, itraconazole [ITZ], GW5074, rupintrivir, and favipiravir) with different mechanisms of action to test their abilities to inhibit EV-A71 replication alone and in combination. All selected compounds have anti-EV-A71 activities in cell culture. The combination of rupintrivir and ITZ or favipiravir was synergistic, while the combination of rupintrivir and suramin was additive. The combination of suramin and favipiravir exerted a strong synergistic antiviral effect. The observed synergy was not due to cytotoxicity, as there was no significant increase in cytotoxicity when compounds were used in combinations at the tested doses. To investigate the potential inhibitory mechanism of favipiravir against enterovirus, two favipiravir-resistant EV-A71 variants were independently selected, and both of them carried an S121N mutation in the finger subdomain of the 3D polymerase. Reverse engineering of this 3D S121N mutation into an infectious clone of EV-A71 confirmed the resistant phenotype. Moreover, viruses resistant to ITZ or favipiravir remained susceptible to other inhibitors. Most notably, combined with ITZ, rupintrivir prevented the development of ITZ-resistant variants. Taken together, these results provide a rational basis for the design of combination regimens for use in the treatment of EV-A71 infections.

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Eupafolin and Ethyl Acetate Fraction ofKalanchoe gracilisStem Extract Show Potent Antiviral Activities against Enterovirus 71 and Coxsackievirus A16

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/591354 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Ching-Ying Wang ◽

Shun-Chueh Huang ◽

Zhen-Rung Lai ◽

Yu-Ling Ho ◽

Yu-Jen Jou ◽

...

Keyword(s):

Ethyl Acetate ◽

Folk Medicine ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Ethyl Acetate Fraction ◽

Coxsackievirus A16 ◽

Aqueous Fraction ◽

Antiviral Activities ◽

Clinical Potential

Enterovirus 71 (EV71) and coxsackievirus A16 (CoxA16) are main pathogens of hand-foot-and-mouth disease, occasionally causing aseptic meningitis and encephalitis in tropical and subtropical regions.Kalanchoe gracilis,Da-Huan-Hun, is a Chinese folk medicine for treating pain and inflammation, exhibiting antioxidant and anti-inflammatory activities. Our prior report (2012) citedK. gracilisleaf extract as moderately active against EV71 and CoxA16. This study further rates antienteroviral potential ofK. gracilisstem (KGS) extract to identify potent antiviral fractions and components. The extract moderately inhibits viral cytopathicity and virus yield, as well asin vitroreplication of EV71 (IC50= 75.18 μg/mL) and CoxA16 (IC50= 81.41 μg/mL). Ethyl acetate (EA) fraction of KGS extract showed greater antiviral activity than that ofn-butanol or aqueous fraction: IC50values of 4.21 μg/mL against EV71 and 9.08 μg/mL against CoxA16. HPLC analysis, UV-Vis absorption spectroscopy, and plaque reduction assay indicate that eupafolin is a vital component of EA fraction showing potent activity against EV71 (IC50= 1.39 μM) and CoxA16 (IC50= 5.24 μM). Eupafolin specifically lessened virus-induced upregulation of IL-6 and RANTES by inhibiting virus-induced ERK1/2, AP-1, and STAT3 signals. Anti-enteroviral potency of KGS EA fraction and eupafolin shows the clinical potential against EV71 and CoxA16 infection.

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Study the current scenario of hand foot mouth disease an Indian prospective

International Journal of Contemporary Pediatrics ◽

10.18203/2349-3291.ijcp20202616 ◽

2020 ◽

Vol 7 (7) ◽

pp. 1558

Author(s):

Ravi Sahota ◽

Navpreet Kaur ◽

Gurpal Singh ◽

Nisha Upadhyay

Keyword(s):

Communicable Disease ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Neurological Complications ◽

Mouth Disease ◽

Human Enterovirus ◽

Cross Sectional ◽

Timely Initiation ◽

Human Enterovirus 71 ◽

Hand Foot Mouth Disease

Background: The hand-foot-mouth disease (HFMD) is an acute communicable disease, mostly affecting children under 5 years of age and caused by human enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16). The usual incubation period is 3 to 7 days. Early symptoms are likely to be fever often followed by a sore throat followed by loss of appetite and general malaise. Aim and objectives was to study the trend of hand foot and mouth disease in a private hospital in Uttarakhand over 5 successive years.Methods: This cross-sectional study was carried among 297 cases of HFMD newborn screened at pediatrics department of Sahota Super-specialty hospital, Kashipur, Uttarakhand during year 2015 to 2019 after ethical clearance of institutional ethical committee. Diagnosis is coded with ICD-10. SPSS version 20 was used to calculate frequencies and percentiles.Results: Almost 29 cases of HMFD were picked in 2015, 32 cases in 2016, 43 cases in 2017, 81 cases in 2018, 112 in 2019. Fever observed in 86% cases. Neurological complications were observed in 9 (3%) cases, pneumonitis in 14 (4.7%) cases, cardiomyopathy observed in 3 (<1%) case. One death was reported.Conclusions: It is vital to screen patients with HFMD for these abnormal clinical presentations, allowing timely initiation of appropriate interventions to reduce the mortality. Increased awareness about vaccination in a developing nation like India and vaccination program at the grass root levels have eradicated certain lethal diseases.

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Functional Insights into Silymarin as an Antiviral Agent against Enterovirus A71 (EV-A71)

International Journal of Molecular Sciences ◽

10.3390/ijms22168757 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8757

Author(s):

Salima Lalani ◽

Malihe Masomian ◽

Chit Laa Poh

Keyword(s):

Foot And Mouth Disease ◽

Antiviral Agent ◽

Underlying Mechanism ◽

Neurological Complications ◽

In Vivo Evaluation ◽

Competitive Binding Assay ◽

Enterovirus A71 ◽

Viral Protein 1

Enterovirus A71 (EV-A71) is a major neurovirulent agent capable of causing severe hand, foot and mouth disease (HFMD) associated with neurological complications and death. Currently, no FDA-approved antiviral is available for the treatment of EV-A71 infections. The flavonoid silymarin was shown to exert virucidal effects, but the binding site on the capsid was unknown. In this study, the ligand interacting site of silymarin was determined in silico and validated in vitro. Moreover, the potential of EV-A71 to develop resistance against silymarin was further evaluated. Molecular docking of silymarin with the capsid of EV-A71 indicated that silymarin binds to viral protein 1 (VP1) of EV-A71, specifically at the GH loop of VP1. The in vitro binding of silymarin with VP1 of EV-A71 was validated using recombinant VP1 through ELISA competitive binding assay. Continuous passaging of EV-A71 in the presence of silymarin resulted in the emergence of a mutant carrying a substitution of isoleucine by threonine (I97T) at position 97 of the BC loop of EV-A71. The mutation was speculated to overcome the inhibitory effects of silymarin. This study provides functional insights into the underlying mechanism of EV-A71 inhibition by silymarin, but warrants further in vivo evaluation before being developed as a potential therapeutic agent.

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Synthesis and Broad Antiviral Activity of Novel 2-aryl-isoindolin-1-ones towards Diverse Enterovirus A71 Clinical Isolates

Molecules ◽

10.3390/molecules24050985 ◽

2019 ◽

Vol 24 (5) ◽

pp. 985 ◽

Cited By ~ 1

Author(s):

Yixuan Wang ◽

Huiqiang Wang ◽

Xinbei Jiang ◽

Zhi Jiang ◽

Tingting Guo ◽

...

Keyword(s):

Antiviral Activity ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Viral Disease ◽

Clinical Isolates ◽

Dependent Manner ◽

Potent Antiviral Activity ◽

Sar Studies ◽

Enterovirus A71

Enterovirus 71 (EV-A71) is the main causative pathogen of childhood hand, foot and mouth disease. Effective medicine is currently unavailable for the treatment of this viral disease. Using the fragment-hopping strategy, a series of 2-aryl-isoindolin-1-one compounds were designed, synthesized and investigated for their in vitro antiviral activity towards multiple EV-A71 clinical isolates (H, BrCr, Shenzhen98, Jiangsu52) in Vero cell culture in this study. The structure–activity relationship (SAR) studies identified 2-phenyl-isoindolin-1-ones as a new potent chemotype with potent antiviral activity against EV-A71. Ten out of the 24 tested compounds showed significant antiviral activity (EC50 < 10 µM) towards four EV-A71 strains. Compounds A3 and A4 exhibited broad and potent antiviral activity with the 50% effective concentration (EC50) values in the range of 1.23–1.76 μM. Moreover, the selectivity indices of A3 and A4 were significantly higher than those of the reference compound, pirodavir. The western blotting experiment indicated that the viral VP1 was significantly decreased at both the protein and RNA level in a dose-dependent manner following treatment with compound A3. Moreover, compound A3 inhibited the viral replication by acting on the virus entry stage. In summary, this study led to the discovery of 2-aryl-isoindolin-1-ones as a promising scaffold with potent anti-EV-A71 activities, which deserves further in-depth studies.

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Formaldehyde-Inactivated Whole-Virus Vaccine Protects a Murine Model of Enterovirus 71 Encephalomyelitis against Disease

Journal of Virology ◽

10.1128/jvi.00999-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 661-665 ◽

Cited By ~ 97

Author(s):

Kien Chai Ong ◽

Shamala Devi ◽

Mary Jane Cardosa ◽

Kum Thong Wong

Keyword(s):

In Situ Hybridization ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Neurological Complications ◽

Viral Culture ◽

Adult Mice ◽

Therapeutic Drugs ◽

Immunohistochemistry Analysis ◽

Old Mice

ABSTRACT Enterovirus 71 (EV71) causes childhood hand, foot, and mouth disease and neurological complications, and no vaccines or therapeutic drugs are currently available. Formaldehyde-inactivated whole-virus vaccines derived from EV71 clinical isolates and a mouse-adapted virus (MAV) were tested in a mouse model of EV71 encephalomyelitis. After only two immunizations, given to mice at 1 and 7 days of age, the MAV vaccine protected mice at 14 days of age from disease. Tissues from immunized mice were negative for virus by viral culture, reverse transcriptase PCR, immunohistochemistry analysis, and in situ hybridization. Cross-neutralizing EV71 antibodies to strains with genotypes B3, B4, and C1 to C5 generated in immunized adult mice were able to passively protect 14-day-old mice from disease.

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