Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal meningoencephalitis complicated with neurogenic pulmonary oedema. Its pathogenesis, especially the CNS involvement, is not clearly understood. The aim of this study was to set up a mouse EV71 infection model with CNS involvement. EV71 virus was administrated orally to neonatal mice. The EV71-infected mice manifested a skin rash at an early stage and hind limb paralysis or death at a later stage. Immunohistochemical staining and virus isolation demonstrated that EV71 replicated in the small intestine, induced viraemia and spread to various organs. Kinetic studies showed that EV71 antigen was first detected in the intestine at 6 h, in the thoracic spinal cord at 24 h, in the cervical spinal cord at 50 h and in the brain stem at 78 h post-infection. Leukocyte infiltration was evident in the spinal cord and brain stem. Furthermore, EV71 virus could be transmitted to littermates within the same cage.
Enterovirus 71 (EV71), a single-positive-stranded RNA virus that belongs to the Enterovirus genus of the Picornaviridae family, is a highly neurotropic virus and has been regarded as the most important neurotropic EV after the eradication of the poliovirus (11). The brain stem is most likely the major target of EV71 infection (2, 8). However, neither the host cell receptor nor the neurotransmission route of EV71 is fully defined.As for poliovirus (PV), two possible routes by which the virus reaches the central nervous system (CNS) have been suggested: the virus either enters the CNS from the blood across the blood-brain barrier (BBB) or is transmitted to the CNS through peripheral nerves via retrograde axonal transport (1,3,14,23). Expression of certain gene segments would be responsible for determining the capacity of PV to spread to the CNS through bloodstream or neuronal pathways (18).In a cynomologus monkey model (12, 13), EV71 showed a wider-spread distribution pattern in the CNS than PV following both intracranial (i.c.) and intravenous inoculations, and monkeys exhibited extrapyramidal signs, including tremor and ataxia. However, after intraspinal inoculation, monkeys developed flaccid paralysis, a pyramidal sign suggesting direct virus invasion in the inoculation site. EV infection and persistence have been implicated in the pathogenesis of certain chronic muscle diseases (17). Furthermore, PV replicated in muscle cells that maintained constant viremia and spread to CNS from peripheral nerves (23). We previously showed that EV71 propagated more effectively in RD (human rhabdomyosarcoma) cells than in SK-N-SH (neuroblastoma) cells and Caco-2 (colorectal adenocarcinoma) cells. Furthermore, after oral (p.o.) inoculation of a mouse-adapted EV71 strain, EV71/MP4, 7-day-old ICR mice developed paralysis, with a mortality rate of 80%. Virus was first seen in the intestine. The virus then spread to muscle and CNS. A vast amount of virus was detected in the CNS and muscle, which led to neuronal loss and rhabdomyolysis of the mice with severe paralysis (21). In this study, we demonstrated that EV71 possesses strong neurotropism and that retrograde axonal transport in neuron cells might represent the major transmission route of EV71 in mice. MATERIALS AND METHODSCells and virus. RD cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum plus 2 mM L-glutamine, 100 IU penicillin, and 100 g of streptomycin per ml. EV71/MP4 strain, a mouse-adapted strain derived from parental virus EV71/Tainan/4643/98 (GenBank accession number AF304458) (22), was grown in RD cells. Working stocks contained 2 ϫ 10 7 PFU/ml. Experimental infection. Specific-pathogen-free, 7-day-old ICR mice
In this study, the contribution of type I interferons (IFNs) to protection against infection with enterovirus 71 (EV71) was investigated using a murine model where the virus was administrated to neonatal Institute of Cancer Research (ICR) mice by either the intraperitoneal (i.p.) or the oral route. In i.p. inoculated mice, post-infection treatment of dexamethasone (5 mg kg−1 at 2 or 3 days after infection) exacerbated clinical symptoms and increased the tissue viral titre. In contrast, polyriboinosinic : polyribocytidylic acid [poly(I : C); 10 or 100 μg per mouse at 12 h before infection], a potent IFN inducer, improved the survival rate and decreased the tissue viral titres after EV71 challenge, which correlated with an increase in serum IFN-α concentration, the percentage of dendritic cells, their expression of major histocompatibility complex class II molecule and IFN-α in spleen. Treatment with a neutralizing antibody for type I IFNs (104 neutralizing units per mouse, 6 h before and 12 h after infection) resulted in frequent deaths and higher tissue viral load in infected mice compared with control mice. In contrast, an early administration of recombinant mouse IFN-αA (104 U per mouse for 3 days starting at 0, 1 or 3 days after infection) protected the mice against EV71 infection. In vitro analysis of virus-induced death in three human cell lines showed that human type I IFNs exerted a direct protective effect on EV71. It was concluded that type I IFNs play an important role in controlling EV71 infection and replication.
Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2(149) (VP2(149M)) or glutamine to glutamic acid substitution at VP1(145) (VP1(145E)) showed greater viral titers and apoptosis. Synergistic effect of VP2(149M) and VP1(145E) double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2(149M) and VP1(145E) mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.
Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase, followed by immunoprecipitation with EV71 particles. Among the 16 proteins identified by tandem mass spectrometry analysis, cell surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens. IMPORTANCEOutbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering the EV71 infection mechanism and anti-EV71 drug discovery but also provide a new strategy for virus receptor identification.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.