A Non-Mouse-Adapted Enterovirus 71 (EV71) Strain Exhibits Neurotropism, Causing Neurological Manifestations in a Novel Mouse Model of EV71 Infection

ABSTRACT Enterovirus 71 (EV71) can cause a severe hand-foot-mouth disease in children. However, the precise mechanism of EV71-associated disease, particularly the neuropathogenesis and pulmonary disorder, is still not fully understood because no suitable animal models are available. The human scavenger receptor class B, member 2 (hSCARB2), is a cellular receptor for EV71. Here, we generated a novel knock-in (KI) mouse model using the CRISPR/Cas9 system to insert the hSCARB2 gene into the mouse Rosa26 locus to study the pathogenesis of EV71. The hSCARB2 KI mice infected with clinical isolates of EV71 showed neurological symptoms, such as ataxia, paralysis, and death. Viral replication was detected in mainly astrocytes and a limited number of neurons and microglia, accompanied by gliosis. Vascular leakage and alveoli filled with erythrocytes were detected, suggesting that edema and hemorrhage, which are observed in human patients, also occurred in EV71-infected KI mice. In addition, proinflammatory cytokines and chemokines were significantly increased in the serum of infected KI mice. These pathological features of the KI mice after infection resembled those of EV71 encephalomyelitis in humans. Therefore, our KI mouse model is suitable to study the pathogenesis of EV71 and is of great significance for development of antiviral drugs and vaccines to treat or prevent EV71 infection. IMPORTANCE Enterovirus 71 (EV71) is associated with severe hand-foot-mouth disease. Recently, outbreaks of EV71 infection with high mortality have been reported in the Asia-Pacific region, posing a great challenge for global public health. To date, the precise mechanism of EV71-induced disease, particularly the neuropathogenesis and respiratory disorders, is still not fully understood because no suitable animal models are available. Human scavenger receptor class B, member 2 (hSCARB2), has been identified as a cellular receptor for EV71. Here, we introduce a novel CRISPR/Cas9-mediated hSCARB2 knock-in (KI) mouse model for the study of EV71 pathogenesis, which is of great significance for the development of antiviral drugs and vaccines.

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Enterovirus 71 infection increases expression of interferon-gamma-inducible protein 10 which protects mice by reducing viral burden in multiple tissues

Journal of General Virology ◽

10.1099/vir.0.046383-0 ◽

2013 ◽

Vol 94 (5) ◽

pp. 1019-1027 ◽

Cited By ~ 18

Author(s):

Fang-Hsiu Shen ◽

Chia-Chun Tsai ◽

Li-Chiu Wang ◽

Kung-Chao Chang ◽

Yuk-Ying Tung ◽

...

Keyword(s):

T Cells ◽

Interferon Gamma ◽

Cd8 T Cells ◽

Enterovirus 71 ◽

Gamma Interferon ◽

Ev71 Infection ◽

Asia Pacific ◽

Virus Clearance ◽

Viral Burden ◽

Inducible Protein

Enterovirus 71 (EV71) infection has induced fatal encephalitis in thousands of young children in the Asia–Pacific region over the last decade. EV71 infection continues to cause serious problems in areas with outbreaks, because vaccines and antiviral therapies are not available. Lymphocytes are present in the brains of infected patients and mice, and they protect mice from infection by decreasing the viral burden. The chemokines responsible for recruiting lymphocytes to infected organs are yet to be identified. Among the lymphocyte chemokines detected, high levels of interferon-gamma-inducible protein-10 (IP-10) are found in the plasma and cerebral spinal fluid of patients with brainstem encephalitis as compared with the levels of a monokine induced by gamma interferon (Mig). Using a murine model to investigate the induction of IP-10 by EV71 infection, we observed that EV71 infection significantly enhanced IP-10 protein expression in the serum and brain, with kinetics similar to viral titres in the blood and brain. Brain neurons of infected mice expressed IP-10. Using wild-type mice and IP-10 gene knockout mice to investigate the role of IP-10 in EV71 infection, we found that IP-10 deficiency significantly reduced levels of Mig in serum, and levels of gamma interferon and the number of CD8 T cells in the mouse brain. Absence of IP-10 significantly increased the mortality of infected mice by 45 %, with slow virus clearance in several vital tissues. Our observations are consistent with a model where EV71 infection boosts IP-10 expression to increase gamma interferon and Mig levels, infiltration of CD8 T cells, virus clearance in tissues and the survival of mice.

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Transplantation of Enterovirus 71 Virion Protein Particle Vaccine Protects Against Enterovirus 71 Infection in a Neonatal Mouse Model

Annals of Transplantation ◽

10.12659/aot.924461 ◽

2021 ◽

Vol 26 ◽

Author(s):

Li Lei ◽

Qing Li ◽

Shuhong Xu ◽

Mingyang Tian ◽

Xinghui Zheng ◽

...

Keyword(s):

Mouse Model ◽

Enterovirus 71 ◽

Neonatal Mouse ◽

Virion Protein ◽

Protein Particle ◽

Enterovirus 71 Infection

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Enterovirus 71 Infection Shapes Host T Cell Receptor Repertoire and Presumably Expands VP1-Specific TCRβ CDR3 Cluster

Pathogens ◽

10.3390/pathogens9020121 ◽

2020 ◽

Vol 9 (2) ◽

pp. 121

Author(s):

Yu-Wen Liao ◽

Bing-Ching Ho ◽

Min-Hsuan Chen ◽

Sung-Liang Yu

Keyword(s):

Central Nervous System ◽

Nervous System ◽

T Cell ◽

T Cell Receptor ◽

Enterovirus 71 ◽

Cell Receptor ◽

Ev71 Infection ◽

Asia Pacific ◽

Binding Prediction ◽

Tcr Repertoire

Enterovirus 71 (EV71) has become an important public health problem in the Asia-Pacific region in the past decades. EV71 infection might cause neurological and psychiatric complications and even death. Although an EV71 vaccine has been currently approved, there is no effective therapy for treating EV71-infected patients. Virus infections have been reported to shape host T cell receptor (TCR) repertoire. Therefore, understanding of host TCR repertoire in EV71 infection could better the knowledge in viral pathogenesis and further benefit the anti-viral therapy development. In this study, we used a mouse-adapted EV71 (mEV71) model to observe changes of host TCR repertoire in an EV71-infected central nervous system. Neonate mice were infected with mEV71 and mouse brainstem TCRβ repertoires were explored. Here, we reported that mEV71 infection impacted host brainstem TCRβ repertoire, where mEV71 infection skewed TCRβ diversity, changed VJ combination usages, and further expanded specific TCRβ CDR3 clones. Using bioinformatics analysis and ligand-binding prediction, we speculated the expanded TCRβ CDR3 clone harboring CASSLGANSDYTF sequence was capable of binding cleaved EV71 VP1 peptides in concert with major histocompatibility complex (MHC) molecules. We observed that mEV71 infection shaped host TCRβ repertoire and presumably expanded VP1-specific TCRβ CDR3 in mEV71-infected mouse brainstem that integrated EV71 pathogenesis in central nervous system.

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Enterovirus 71 Represses Interleukin Enhancer-Binding Factor 2 Production and Nucleus Translocation to Antagonize ILF2 Antiviral Effects

Viruses ◽

10.3390/v12010022 ◽

2019 ◽

Vol 12 (1) ◽

pp. 22

Author(s):

Jing Jin ◽

Wenbiao Wang ◽

Sha Ai ◽

Weiyong Liu ◽

Yu Song ◽

...

Keyword(s):

Nonstructural Protein ◽

Enterovirus 71 ◽

Ev71 Infection ◽

Health It ◽

Antiviral Effects ◽

Distinct Mechanism ◽

Binding Factor ◽

2B Protein ◽

Rd Cells ◽

Hand Foot Mouth Disease

Enterovirus 71 (EV71) infection causes hand-foot-mouth disease (HFMD), meningoencephalitis, neonatal sepsis, and even fatal encephalitis in children, thereby presenting a serious risk to public health. It is important to determine the mechanisms underlying the regulation of EV71 infection. In this study, we initially show that the interleukin enhancer-binding factor 2 (ILF2) reduces EV71 50% tissue culture infective dose (TCID50) and attenuates EV71 plaque-formation unit (PFU), thereby repressing EV71 infection. Microarray data analyses show that ILF2 mRNA is reduced upon EV71 infection. Cellular studies indicate that EV71 infection represses ILF2 mRNA expression and protein production in human leukemic monocytes (THP-1) -differentiated macrophages and human rhabdomyosarcoma (RD) cells. In addition, EV71 nonstructural protein 2B interacts with ILF2 in human embryonic kidney (HEK293T) cells. Interestingly, in the presence of EV71 2B, ILF2 is translocated from the nucleus to the cytoplasm, and it colocalizes with 2B in the cytoplasm. Therefore, we present a distinct mechanism by which EV71 antagonizes ILF2-mediated antiviral effects by inhibiting ILF2 expression and promoting ILF2 translocation from the nucleus to the cytoplasm through its 2B protein.

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Hypoxia and therapeutic treatment of EV-A71 with an immune modulator TLR7 agonist in a new immunocompetent mouse model

Journal of Biomedical Science ◽

10.1186/s12929-019-0585-y ◽

2019 ◽

Vol 26 (1) ◽

Author(s):

An-Ting Liou ◽

Chun-Che Liao ◽

Shu-Fan Chou ◽

Ya-Shu Chang ◽

Chih-Shin Chang ◽

...

Keyword(s):

Mouse Model ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Research Direction ◽

Oral Route ◽

Drug Candidate ◽

Immunocompetent Mouse ◽

Immune Modulators ◽

Immune Modulator ◽

New Research

Abstract Background Enterovirus 71 (EV71 or EV-A71) was first identified in California about half a century ago. In recent years, outbreaks of EV-A71 were prevalent worldwide, including Taiwan, Malaysia, Singapore, Japan, and China. Between 2008 and 2011, China alone reported 1894 deaths associated with EV-A71 infection. In mild cases, EV-A71 can cause herpangina and hand-foot-and-mouth disease (HFMD). However, in severe cases, it could cause neurological disorders, including meningitis and encephalitis. Cardiopulmonary failure is common among hospitalized children with EV-A71 infection. No effective FDA-approved therapeutics against EV-A71 are clinically available. Methods We report the establishment of an immunocompetent wild type strain 129 (wt-129) mouse model, which can be cross-species infected with human EV-A71 clinical isolates via an intraperitoneal route. Results One intriguing disease phenotype of this new model is the development of characteristic “White-Jade” patches in the muscle, which lost sporadically the normal pink color of uninfected muscle. Viral VP1 protein and massive leukocyte infiltration were detected in muscles with or without white-jades. We demonstrated further that hypoxia is a general phenomenon associated with white-jades in both immunocompetent and immunodeficient mouse models. Therefore, hypoxia appears to be a feature intrinsic to EV-A71 infection, irrespective of its host’s immunogenetic background. To date, no effective treatment for EV-A71 is available. Here, using this new wt-129 mouse model, we showed that timely treatment with compound R837 (a TLR7 immune modulator) via oral or intraperitoneal routes, rescued the hypoxia, limb paralysis, and death at a high therapeutic efficacy. Conclusions In this new immunocompetent mouse 129 model, we observed an unexpected white-jade phenotype and its associated hypoxia. The successful treatment with TLR7 immune modulators via an oral route, provide us a new research direction for EV-A71 basic science and translational research. It remains an open issue whether R837 or its related compounds, will be a promising drug candidate in clinical trials in EV-A71 endemic or epidemic areas in the future.

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Plant-Produced Anti-Enterovirus 71 (EV71) Monoclonal Antibody Efficiently Protects Mice Against EV71 Infection

Plants ◽

10.3390/plants8120560 ◽

2019 ◽

Vol 8 (12) ◽

pp. 560 ◽

Cited By ~ 5

Author(s):

Kaewta Rattanapisit ◽

Zhang Chao ◽

Konlavat Siriwattananon ◽

Zhong Huang ◽

Waranyoo Phoolcharoen

Keyword(s):

Monoclonal Antibody ◽

Therapeutic Option ◽

Enterovirus 71 ◽

Cost Effective ◽

Ev71 Infection ◽

Asia Pacific ◽

Variable Regions ◽

Hand Foot Mouth Disease ◽

Protection Against Infection

Enterovirus 71 (EV71) is the main causative agent of severe hand-foot-mouth disease. EV71 affects countries mainly in the Asia-Pacific region, which makes it unattractive for pharmaceutical companies to develop drugs or vaccine to combat EV71 infection. However, development of these drugs and vaccines is vital to protect younger generations. This study aims to develop a specific monoclonal antibody (mAb) to EV71 using a plant platform, which is a cost-effective and scalable production technology. A previous report showed that D5, a murine anti-EV71 mAb, binds to VP1 protein of EV71, potently neutralizes EV71 in vitro, and effectively protects mice against EV71 infection. Herein, plant-produced chimeric D5 (cD5) mAb, variable regions of murine D5 antibody linked with constant regions of human IgG1, was transiently expressed in Nicotiana benthamiana using geminiviral vectors. The antibody was expressed at high levels within six days of infiltration. Plant-produced cD5 retained its in vitro high-affinity binding and neutralizing activity against EV71. Furthermore, a single dose (10 µg/g body weight) of plant-produced cD5 mAb offered 100% protection against infection in mice after a lethal EV71 challenge. Therefore, our results showed that plant-produced anti-EV71 mAb is an effective, safe, and affordable therapeutic option against EV71 infection.

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Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax

Journal of Virology ◽

10.1128/jvi.01499-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 9862-9877 ◽

Cited By ~ 22

Author(s):

Haolong Cong ◽

Ning Du ◽

Yang Yang ◽

Lei Song ◽

Wenliang Zhang ◽

...

Keyword(s):

Cell Apoptosis ◽

Enterovirus 71 ◽

Terminal Region ◽

Ev71 Infection ◽

Apoptotic Pathway ◽

Hydrophobic Region ◽

Mitochondrial Apoptotic Pathway ◽

Proapoptotic Protein ◽

2B Protein ◽

Induced Apoptosis

ABSTRACTTo survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B-expressing cells that were treated with a recombinant Bax showed increased Bax interaction and cytochromec(Cytc) release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax knockdown cells but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71- or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal region of 2B (aa 63 to 80) was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, overexpression of the antiapoptotic protein Bcl-XLabrogates 2B-induced release of Cytcand caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of proapoptotic protein Bax.IMPORTANCEEV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage is a possible process of EV71-related pathogenesis. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. This study provides evidence that EV71 induces cell apoptosis by modulating Bax activation and reveals important clues regarding the mechanism of Cytcrelease and mitochondrial permeabilization during EV71 infection.

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