scholarly journals Susceptibility and cytokine responses of human neuronal cells to multiple circulating EV-A71 genotypes in India

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Madhu Chhanda Mohanty ◽  
Swapnil Yashavant Varose ◽  
Vinay Kumar Saxena
Keyword(s):  
Inflammatory Cytokines ◽  
Neuronal Cells ◽  
Foot And Mouth Disease ◽  
Asia Pacific ◽  
Delayed Response ◽  
Cytokine Responses ◽  
Enterovirus A71 ◽  
Infected Cells ◽  
Human Neuronal Cells

AbstractEnterovirus-A71 (EV-A71) associated Hand, foot and mouth disease (HFMD) is a highly contagious viral infection affecting children in Asia–Pacific region and has become a major threat to public health. Although several EV-A71 genotypes (C, D, and G) were isolated in India in recent years, no recognizable outbreak of EV-A71 caused HFMD, Acute Flaccid paralysis (AFP) or encephalitis have been reported so far. It is essential to study the pathogenicity or cell tropism of these Indian isolates in order to understand their tendency to cause disease. We investigated the susceptibility and cytokine responses of indigenous EV-A71 genotypes (D and G) isolated from cases of AFP and genotype C viruses isolated from cases of HFMD and encephalitis, in human cells in-vitro. Although all three EV-A71 genotypes could infect and replicate in human muscle and neuronal cells, the genotype D virus showed a delayed response in human neuronal cells. Quantification of cytokine secretion in response to these isolates followed by confirmation with gene expression assays in human neuronal cells revealed significantly higher secretion of pro-inflammatory cytokines TNF-α IL-8, IL-6, IP-10 (p < 0.001) in G genotype infected cells as compared to pathogenic C genotypes whereas the genotype D virus could not induce any of the inflammatory cytokines. These findings will help to better understand the host response to indigenous EV-A71 genotypes for management of future EV-A71 outbreaks in India, if any.

Enterovirus Diseases and Vaccines

2021 ◽  
Author(s):  
Heinz-Josef Schmitt
Keyword(s):  
Disease Outbreaks ◽  
Foot And Mouth Disease ◽  
Asia Pacific ◽  
Asia Pacific Region ◽  
Internal Organs ◽  
Enterovirus A71 ◽  
Life Threatening ◽  
Development Pipeline ◽  
The Brain ◽  
Genus Enterovirus

Enterovirus A71 (EV A71) (genus enterovirus, family pircornaviridae) causes benign vesicular lesions on skin (hand, foot and mouth disease, HFMD) and mucous membranes of the mouth (herpangina), and also severe to life-threatening infections of the brain, the heart, and other internal organs. Disease outbreaks in the Asia-Pacific region regularly involve thousands of children <5 years resulting in many deaths. Such outbreaks are caused by specific EV genotypes that vary by time and place. While there are various promising and innovative options for treatment in development, none are licensed to date. Immunoglobulins may be beneficial through virus neutralization and modulation of the inflammatory response by the host. In China, 3 different highly efficacious and safe vaccines are commercially available; however, none are licensed outside the country. Roughly half a dozen vaccines are in the development pipeline, with some using innovative approaches and trying to broaden strain coverage.

scholarly journals Molecular epidemiology of an enterovirus A71 outbreak associated with severe neurological disease, Spain, 2016

2019 ◽  
Vol 24 (7) ◽  
Author(s):  
Rubén González-Sanz ◽  
Didac Casas-Alba ◽  
Cristian Launes ◽  
Carmen Muñoz-Almagro ◽  
María Montserrat Ruiz-García ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Phylogenetic Analyses ◽  
Clinical Manifestations ◽  
Foot And Mouth Disease ◽  
Neurological Complications ◽  
Asia Pacific ◽  
Enterovirus A71 ◽  
Hospitalised Patients ◽  
Wide Range ◽  
Large Outbreak

Introduction Enterovirus A71 (EV-A71) is an emerging pathogen that causes a wide range of disorders including severe neurological manifestations. In the past 20 years, this virus has been associated with large outbreaks of hand, foot and mouth disease with neurological complications in the Asia-Pacific region, while in Europe mainly sporadic cases have been reported. In spring 2016, however, an EV-A71 outbreak associated with severe neurological cases was reported in Catalonia and spread further to other Spanish regions. Aim Our objective was to investigate the epidemiology and clinical characteristics of the outbreak. Methods We carried out a retrospective study which included 233 EV-A71-positive samples collected during 2016 from hospitalised patients. We analysed the clinical manifestations associated with EV-A71 infections and performed phylogenetic analyses of the 3’-VP1 and 3Dpol regions from all Spanish strains and a set of EV-A71 from other countries. Results Most EV-A71 infections were reported in children (mean age: 2.6 years) and the highest incidence was between May and July 2016 (83%). Most isolates (218/233) were classified as subgenogroup C1 and 217 of them were grouped in one cluster phylogenetically related to a new recombinant variant strain associated with severe neurological diseases in Germany and France in 2015 and 2016. Moreover, we found a clear association of EV-A71-C1 infection with severe neurological disorders, brainstem encephalitis being the most commonly reported. Conclusion An emerging recombinant variant of EV-A71-C1 was responsible for the large outbreak in 2016 in Spain that was associated with many severe neurological cases.

Virulence of enterovirus-A71 fluctuates depending on the phylogenetic clade formed in the epidemic year and epidemic region

2021 ◽  
Author(s):  
Kyousuke Kobayashi ◽  
Hidekazu Nishimura ◽  
Katsumi Mizuta ◽  
Tomoha Nishizawa ◽  
Son T. Chu ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Foot And Mouth Disease ◽  
Virus Genome ◽  
Mouth Disease ◽  
Asia Pacific ◽  
Enterovirus A71 ◽  
Viral Virulence ◽  
Foot And Mouth ◽  
Potential Risks ◽  
A Minor

Although epidemics of hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) have occurred worldwide, the Asia-Pacific region has seen large sporadic outbreaks with many severe neurological cases. This suggests that the virulence of the circulating viruses fluctuates in each epidemic and that HFMD outbreaks with many severe cases occur when highly virulent viruses are circulating predominantly, which has not been experimentally verified. Here, we analyzed 32 clinically isolated strains obtained in Japan from 2002 to 2013, along with 27 Vietnamese strains obtained from 2015 to 2016 that we characterized previously using human SCARB2 transgenic mice. Phylogenetic analysis of the P1 region classified them into five clades belonging to subgenogroup B5 (B5-I to B5-V) and five clades belonging to subgenogroup C4 (C4-I to C4-V) according to the epidemic year and region. Interestingly, the clade B5-I and B5-II were very virulent, while clades B5-III, B5-IV, and B5-V were less virulent. Clades C4-II, C4-III, C4-IV, and C4-V were virulent, while clade C4-I was not. The result experimentally showed for the first time that several clades with different virulence levels emerged one after another. The experimental virulence evaluation of circulating viruses using SCARB2 transgenic mice is helpful to assess potential risks of circulating viruses. These results also suggest that a minor nucleotide or amino acid substitution in the EV-A71 genome during circulation may cause fluctuations in virulence. The data presented herein may increase our understanding of the dynamics of viral virulence during epidemics. IMPORTANCE Outbreaks of hand, foot, and mouth disease (HFMD) with severe enterovirus A71 (EV-A71) cases have occurred repeatedly, mainly in Asia. In severe cases, central nervous system complications can lead to death, making it an infectious disease of importance to public health. An unanswered question about this disease is why outbreaks of HFMD with many severe cases sometimes occur. Here, we collected EV-A71 strains that were prevalent in Japan and Vietnam over the past 20 years and evaluated their virulence in a mouse model of EV-A71 infection. This method clearly revealed that viruses belonging to different clades have different virulence, indicating that the method is powerful to assess the potential risks of the circulating viruses. The results also suggested that factors in the virus genome may potentially cause an outbreak with many severe cases and that further studies may facilitate the prediction of large epidemics of EV-A71 in the future.

scholarly journals Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Gisselle N. Medina ◽  
Paul Azzinaro ◽  
Elizabeth Ramirez-Medina ◽  
Joseph Gutkoska ◽  
Ying Fang ◽  
...  
Keyword(s):  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Type I ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Infected Cells ◽  
Viral Attenuation

ABSTRACT Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD. IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD.

scholarly journals RSAD2 and AIM2 Modulate Coxsackievirus A16 and Enterovirus A71 Replication in Neuronal Cells in Different Ways That May Be Associated with Their 5′ Nontranslated Regions

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Thinesshwary Yogarajah ◽  
Kien Chai Ong ◽  
David Perera ◽  
Kum Thong Wong
Keyword(s):  
Viral Replication ◽  
Neuronal Cells ◽  
Neuroblastoma Cells ◽  
Foot And Mouth Disease ◽  
Neurological Complications ◽  
Mouth Disease ◽  
Host Responses ◽  
Coxsackievirus A16 ◽  
Enterovirus A71 ◽  
Foot And Mouth

ABSTRACT Coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) are closely related enteroviruses that cause the same hand, foot, and mouth disease (HFMD), but neurological complications occur only very rarely in CV-A16 compared to EV-A71 infections. To elucidate host responses that may be able to explain these differences, we performed transcriptomic analysis and real-time quantitative PCR (RT-qPCR) in CV-A16-infected neuroblastoma cells (SK-N-SH), and the results showed that the radical S -adenosylmethionine domain containing 2 (RSAD2) was the highest upregulated gene in the antimicrobial pathway. Increased RSAD2 expression was correlated with reduced viral replication, while RSAD2 knockdown cells were correlated with increased replication. EV-A71 replication showed no apparent correlation to RSAD2 expressions. Absent in melanoma 2 (AIM2), which is associated with pyroptotic cell death, was upregulated in EV-A71-infected neurons but not in CV-A16 infection, suggesting that the AIM2 inflammasome played a significant role in suppressing EV-A71 replication. Chimeric viruses derived from CV-A16 and EV-A71 but containing swapped 5′ nontranslated regions (5′ NTRs) showed that RSAD2 expression/viral replication and AIM2 expression/viral replication patterns may be linked to the 5′ NTRs of parental viruses. Differences in secondary structure of internal ribosomal entry sites within the 5′ NTR may be responsible for these findings. Overall, our results suggest that CV-A16 and EV-A71 elicit different host responses to infection, which may help explain the apparent lower incidence of CV-A16-associated neurovirulence in HFMD outbreaks compared to EV-A71 infection. IMPORTANCE Although coxsackievirus A16 (CV-A16) and enterovirus A17 (EV-A71) both cause hand, foot, and mouth disease, EV-A71 has emerged as a leading cause of nonpolio, enteroviral fatal encephalomyelitis among young children. The significance of our research is in the identification of the possible differing and novel mechanisms of CV-A16 and EV-A71 inhibition in neuronal cells that may impact viral neuropathogenesis. We further showed that viral 5′ NTRs may play significant roles in eliciting different host response mechanisms.

scholarly journals 2-Iminobiotin Superimposed on Hypothermia Protects Human Neuronal Cells from Hypoxia-Induced Cell Damage: An in Vitro Study

2018 ◽  
Vol 8 ◽  
Author(s):  
Karina Zitta ◽  
Cacha Peeters-Scholte ◽  
Lena Sommer ◽  
Matthias Gruenewald ◽  
Lars Hummitzsch ◽  
...  
Keyword(s):  
Cell Damage ◽  
Neuronal Cells ◽  
In Vitro Study ◽  
Vitro Study ◽  
Human Neuronal Cells

scholarly journals Demonstration of Co-Infection and Trans-Encapsidation of Viral RNA In Vitro Using Epitope-Tagged Foot-and-Mouth Disease Viruses

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2433
Author(s):  
Kay Childs ◽  
Nicholas Juleff ◽  
Katy Moffat ◽  
Julian Seago
Keyword(s):  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Flag Epitope ◽  
Experimental Systems ◽  
Infection Dynamics ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Infected Cells ◽  
Vp1 Capsid Protein

Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is an economically devastating disease affecting several important livestock species. FMDV is antigenically diverse and exists as seven serotypes comprised of many strains which are poorly cross-neutralised by antibodies induced by infection or vaccination. Co-infection and recombination are important drivers of antigenic diversity, especially in regions where several serotypes co-circulate at high prevalence, and therefore experimental systems to study these events in vitro would be beneficial. Here we have utilised recombinant FMDVs containing an HA or a FLAG epitope tag within the VP1 capsid protein to investigate the products of co-infection in vitro. Co-infection with viruses from the same and from different serotypes was demonstrated by immunofluorescence microscopy and flow cytometry using anti-tag antibodies. FLAG-tagged VP1 and HA-tagged VP1 could be co-immunoprecipitated from co-infected cells, suggesting that newly synthesised capsids may contain VP1 proteins from both co-infecting viruses. Furthermore, we provide the first demonstration of trans-encapsidation of an FMDV genome into capsids comprised of proteins encoded by a co-infecting heterologous virus. This system provides a useful tool for investigating co-infection dynamics in vitro, particularly between closely related strains, and has the advantage that it does not depend upon the availability of strain-specific FMDV antibodies.

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

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.

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

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 985 ◽  
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.

scholarly journals A Mouse Model for Infection with Enterovirus A71 in Small Extracellular Vesicles

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Jiaqi Gu ◽  
Jing Wu ◽  
Yuwen Cao ◽  
Xinran Zou ◽  
Xiaonan Jia ◽  
...  
Keyword(s):  
Mouse Model ◽  
Extracellular Vesicles ◽  
Foot And Mouth Disease ◽  
Vital Role ◽  
Infection Model ◽  
Neurogenic Pulmonary Edema ◽  
Enterovirus A71 ◽  
Important Pathway ◽  
Infected Cells ◽  
Circulation Of The Blood

ABSTRACT Enterovirus A71 (EV-A71) is the major pathogen of hand, foot, and mouth disease (HFMD); in some severe cases, it could develop into central nervous system (CNS) disease such as aseptic meningitis, encephalitis, and neurogenic pulmonary edema in children under 5 years. The EV-A71 pathogenesis which is involved with the CNS is unclear due to the lack of a simple and reliable mouse model thus far. Most clinical EV-A71 isolates could not effectively infect the neonatal mouse, which used to be an EV-A71 infection model. The small extracellular vesicles (sEVs) released from clinical EV-A71 isolate-infected cells were infectious in cell lines and could cause a high viral replication in mice. Neonatal ICR mice were injected intraperitoneally with these infectious sEVs and showed more weight loss and higher mortality than those mice injected with the clinical EV-A71 isolate. By using these sEVs, we provided a simple and effective method by which we can generate a stable and valuable animal model for the studies of EV-A71 pathogenesis and therapy. IMPORTANCE EV-A71 was supposed to infect the CNS through the neural pathway and the circulation of the blood in previous studies. Reverse axon transport had been confirmed as an important pathway for EV-A71 to infect the CNS; however, it is still unknown how EV-A71 infects the CNS through the circulation of the blood. Combined with the infectivity of sEVs secreted from EV-A71-infected cells and the characteristic that sEVs could cross the blood-brain barrier, we considered that sEVs may play a vital role in EV-A71 pathogenesis of the CNS.

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