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The Preferential Infection of Astrocytes by Enterovirus 71 Plays a Key Role in the Viral Neurogenic Pathogenesis.

ABSTRACT: The pathological manifestations of fatal cases of human hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) are characterized by inflammatory damage to the central nervous system (CNS). Here, the dynamic distribution of EV71 in the CNS and the subsequent pathological characteristics within different regions of neonatal rhesus macaque brain tissue were studied using a chimeric EV71 expressing green fluorescence protein. The results were compared with brain tissue obtained from the autopsies of deceased EV71-infected HFMD patients. These observations suggested that the virus was prevalent in areas around the blood vessels and nerve nuclei in the brain stem and showed a preference for astrocytes in the CNS. Interestingly, infected astrocytes within the in vivo and in vitro human and macaque systems exhibited increased expression of excitatory neurotransmitters and cytokines that also stimulated the neuronal secretion of the excitatory neurotransmitters noradrenalin and adrenalin, and this process most likely plays a role in the pathophysiological events that occur during EV71 infection.

SUBMITTER: Feng M

PROVIDER: S-EPMC5174126 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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The Preferential Infection of Astrocytes by Enterovirus 71 Plays a Key Role in the Viral Neurogenic Pathogenesis.

Feng Min M Guo Sujie S Fan Shengtao S Zeng Xiaofeng X Zhang Ying Y Liao Yun Y Wang Jianbin J Zhao Ting T Wang Lichun L Che Yanchun Y Wang Jingjing J Ma Na N Liu Longding L Yue Lei L Li Qihan Q

Frontiers in cellular and infection microbiology 20161221

The pathological manifestations of fatal cases of human hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) are characterized by inflammatory damage to the central nervous system (CNS). Here, the dynamic distribution of EV71 in the CNS and the subsequent pathological characteristics within different regions of neonatal rhesus macaque brain tissue were studied using a chimeric EV71 expressing green fluorescence protein. The results were compared with brain tissue obtained from th ...[more]

PMID: 28066727

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Project description:BackgroundWe previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.ResultsWe demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH₄Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.ConclusionsIn this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.

Project description:A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications.

Project description:AimTo characterise the viral kinetics of enterovirus 71 (EV71).MethodsIn this study, human rhabdomyosarcoma (RD) cells were infected with EV71 at different multiplicity of infection (MOI). After infection, the cytopathic effect (CPE) was monitored and recorded using a phase contrast microscope associated with a CCD camera at different time points post viral infection (0, 6, 12, 24 h post infection). Cell growth and viability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in both EV71 infected and mock infected cells at each time point. EV71 replication kinetics in RD cells was determined by measuring the total intracellular viral RNA with real-time reverse-transcription polymerase chain reaction (qRT-PCR). Also, the intracellular and extracellular virion RNA was isolated and quantified at different time points to analyze the viral package and secretion. The expression of viral protein was determined by analyze the levels of viral structure protein VP1 with Western blotting.ResultsEV71 infection induced a significant CPE as early as 6 h post infection (p.i.) in both RD cells infected with high ratio of virus (MOI 10) and low ratio of virus (MOI 1). In EV71 infected cells, the cell growth was inhibited and the number of viable cells was rapidly decreased in the later phase of infection. EV71 virions were uncoated immediately after entry. The intracellular viral RNA began to increase at as early as 3 h p.i. and the exponential increase was found between 3 h to 6 h p.i. in both infected groups. For viral structure protein synthesis, results from western-blot showed that intracellular viral protein VP1 could not be detected until 6 h p.i. in the cells infected at either MOI 1 or MOI 10; and reached the peak at 9 h p.i. in the cells infected with EV71 at both MOI 1 and MOI 10. Simultaneously, the viral package and secretion were also actively processed as the virus underwent rapid replication. The viral package kinetics was comparable for both MOI 1 and MOI 10 infected groups. It was observed that at 3 h p.i, the intracellular virions obviously decreased, thereafter, the intracellular virions began to increase and enter into the exponential phase until 12 h p.i. The total amounts of intracellular virons were decreased from 12 to 24 h p.i. Consistent with this result, the increase of virus secretion occurred during 6 to 12 h p.i.ConclusionThe viral kinetics of EV71 were established by analyzing viral replication, package and secretion in RD cells.

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The Preferential Infection of Astrocytes by Enterovirus 71 Plays a Key Role in the Viral Neurogenic Pathogenesis.

Publications

The Preferential Infection of Astrocytes by Enterovirus 71 Plays a Key Role in the Viral Neurogenic Pathogenesis.

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