Neurotropism of Enterovirus D68 Isolates Is Independent of Sialic Acid and Is Not a Recently Acquired Phenotype

ABSTRACT Acute flaccid myelitis (AFM) is a rare but serious illness of the nervous system, specifically affecting the gray matter of the spinal cord, motor-controlling regions of the brain, and cranial nerves. Most cases of AFM are pathogen associated, typically with poliovirus and enterovirus infections, and occur in children under the age of 6 years. Enterovirus D68 (EV-D68) was first isolated from children with pneumonia in 1962, but an association with AFM was not observed until the 2014 outbreak. Organotypic mouse brain slice cultures generated from postnatal day 1 to 10 mice and adult ifnar knockout mice were used to determine if neurotropism of EV-D68 is shared among virus isolates. All isolates replicated in organotypic mouse brain slice cultures, and three isolates replicated in primary murine astrocyte cultures. All four EV-D68 isolates examined caused paralysis and death in adult ifnar knockout mice. In contrast, no viral disease was observed after intracranial inoculation of wild-type mice. Six of the seven EV-D68 isolates, including two from 1962 and four from the 2014 outbreak, replicated in induced human neurons, and all of the isolates replicated in induced human astrocytes. Furthermore, a putative viral receptor, sialic acid, is not required for neurotropism of EV-D68, as viruses replicated within neurons and astrocytes independent of binding to sialic acid. These observations demonstrate that EV-D68 is neurotropic independent of its genetic lineage and can infect both neurons and astrocytes and that neurotropism is not a recently acquired characteristic as has been suggested. Furthermore, the results show that in mice the innate immune response is critical for restricting EV-D68 disease. IMPORTANCE Since 2014, numerous outbreaks of childhood infections with enterovirus D68 (EV-D68) have occurred worldwide. Most infections are associated with flu-like symptoms, but paralysis may develop in young children. It has been suggested that infection only with recent viral isolates can cause paralysis. To address the hypothesis that EV-D68 has recently acquired neurotropism, murine organotypic brain slice cultures, induced human motor neurons and astrocytes, and mice lacking the alpha/beta interferon receptor were infected with multiple virus isolates. All EV-D68 isolates, from 1962 to the present, can infect neural cells, astrocytes, and neurons. Furthermore, our results show that sialic acid binding does not play a role in EV-D68 neuropathogenesis. The study of EV-D68 infection in organotypic brain slice cultures, induced motor neurons, and astrocytes will allow for the elucidation of the mechanism by which the virus infection causes disease.

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Neurotropism of enterovirus D68 isolates is independent of sialic acid and is not a recently acquired phenotype

10.1101/161778 ◽

2017 ◽

Author(s):

Amy B Rosenfeld ◽

Audrey L Warren ◽

Vincent R Racaniello

Keyword(s):

Sialic Acid ◽

Brain Slice ◽

Neurological Dysfunction ◽

Genetic Lineage ◽

Viral Receptor ◽

Number Of Children ◽

Acute Flaccid Myelitis ◽

Enterovirus D68 ◽

Organotypic Brain Slice ◽

Brain Slice Cultures

AbstractAcute flaccid myelitis /acute flaccid paralysis (AFM/AFP) is a rare but serious illness of the nervous system, specifically affecting the grey matter of the spinal cord, motor controlling regions of the brain and the cranial nerve. Most cases of AFM/AFP are pathogen associated, typically with poliovirus and enterovirus infections, and occur in children under the age of 6 years old. Enterovirus D68 (EV-D68) was first isolated from children with pneumonia in 1962, but an association with AFM/AFP was not observed until the 2014 outbreak. Organotypic mouse brain slice cultures generated from postnatal day 1 to 10 mice were used to determine if neurotropism of EV-D68 is shared among virus isolates. Six of the seven EV-D68 isolates examined, including two from 1962 and four from the 2014 outbreak, replicated in neurons, and all replicated in astrocytes. Furthermore, a putative viral receptor, sialic acid, is not required for neurotropism of EV-D68, as both sialic acid dependent and independent viruses replicated within neurons. These observations demonstrate that EV-D68 is neurotropic independent of its genetic lineage, can infect both neurons and astrocytes, and that neurotropism is not a recently acquired characteristic as has been suggested.SignificanceRecently there has been an increase in the number of children infected with enterovirus D68 (EV-D68). Most infections are associated with mild flu-like symptoms, but neurological dysfunction may develop in a small number of children. How the biochemical and genetic differences among EV-D68 isolates relates to development of neurological disease remains an unanswered question. Assessing infection of multiple viral isolates in organotypic brain slice cultures from postnatal day 1 to 10 mice revealed that multiple isolates are neurotropic. Both neuraminidase sensitive and resistant viruses infected neurons, indicating that sialic acid binding does not play a role in EV-D68 neuropathogenesis. Establishment of a genetically and pharmacologically amenable system using organotypic brain slice cultures will provide insight into how EV-D68 neuropathologies develop.

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Organotypic brain slice cultures as a model to study angiogenesis of brain vessels

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2015.00052 ◽

2015 ◽

Vol 3 ◽

Cited By ~ 18

Author(s):

Bianca Hutter-Schmid ◽

Kathrin M. Kniewallner ◽

Christian Humpel

Keyword(s):

Brain Slice ◽

Brain Vessels ◽

Organotypic Brain Slice ◽

Brain Slice Cultures

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Preparation of organotypic brain slice cultures for the study of Alzheimer’s disease

F1000Research ◽

10.12688/f1000research.14500.1 ◽

2018 ◽

Vol 7 ◽

pp. 592

Author(s):

Cara L. Croft ◽

Wendy Noble

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Brain Slice ◽

Synaptic Dysfunction ◽

Slice Culture ◽

Therapeutic Effects ◽

Culture Model ◽

Organotypic Brain Slice ◽

Brain Slice Cultures

Alzheimer's disease, the most common cause of dementia, is a progressive neurodegenerative disorder characterised by amyloid-beta deposits in extracellular plaques, intracellular neurofibrillary tangles of aggregated tau, synaptic dysfunction and neuronal death. There are no cures for AD and current medications only alleviate some disease symptoms. Transgenic rodent models to study Alzheimer’s mimic features of human disease such as age-dependent accumulation of abnormal beta-amyloid and tau, synaptic dysfunction, cognitive deficits and neurodegeneration. These models have proven vital for improving our understanding of the molecular mechanisms underlying AD and for identifying promising therapeutic approaches. However, modelling neurodegenerative disease in animals commonly involves aging animals until they develop harmful phenotypes, often coupled with invasive procedures. In vivo studies are also resource, labour, time and cost intensive. We have developed a novel organotypic brain slice culture model to study Alzheimer’ disease which brings the potential of substantially reducing the number of rodents used in dementia research from an estimated 20,000 per year. We obtain 36 brain slices from each mouse pup, considerably reducing the numbers of animals required to investigate multiple stages of disease. This tractable model also allows the opportunity to modulate multiple pathways in tissues from a single animal. We believe that this model will most benefit dementia researchers in the academic and drug discovery sectors. We validated the slice culture model against aged mice, showing that the molecular phenotype closely mimics that displayed in vivo, albeit in an accelerated timescale. We showed beneficial outcomes following treatment of slices with agents previously shown to have therapeutic effects in vivo, and we also identified new mechanisms of action of other compounds. Thus, organotypic brain slice cultures from transgenic mouse models expressing Alzheimer’s disease-related genes may provide a valid and sensitive replacement for in vivo studies that do not involve behavioural analysis.

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Contemporary Circulating Enterovirus D68 Strains Infect and Undergo Retrograde Axonal Transport in Spinal Motor Neurons Independent of Sialic Acid

Journal of Virology ◽

10.1128/jvi.00578-19 ◽

2019 ◽

Vol 93 (16) ◽

Cited By ~ 14

Author(s):

Alison M. Hixon ◽

Penny Clarke ◽

Kenneth L. Tyler

Keyword(s):

Sialic Acid ◽

Motor Neuron ◽

Axonal Transport ◽

Motor Neurons ◽

The United States ◽

Retrograde Axonal Transport ◽

Spinal Motor Neurons ◽

Acute Flaccid Myelitis ◽

Spinal Motor ◽

Enterovirus D68

ABSTRACTEnterovirus D68 (EV-D68) is an emerging virus that has been identified as a cause of recent outbreaks of acute flaccid myelitis (AFM), a poliomyelitis-like spinal cord syndrome that can result in permanent paralysis and disability. In experimental mouse models, EV-D68 spreads to, infects, and kills spinal motor neurons following infection by various routes of inoculation. The topography of virus-induced motor neuron loss correlates with the pattern of paralysis. The mechanism(s) by which EV-D68 spreads to target motor neurons remains unclear. We sought to determine the capacity of EV-D68 to spread by the neuronal route and to determine the role of known EV-D68 receptors, sialic acid and intracellular adhesion molecule 5 (ICAM-5), in neuronal infection. To do this, we utilized a microfluidic chamber culture system in which human induced pluripotent stem cell (iPSC) motor neuron cell bodies and axons can be compartmentalized for independent experimental manipulation. We found that EV-D68 can infect motor neurons via their distal axons and spread by retrograde axonal transport to the neuronal cell bodies. Virus was not released from the axons via anterograde axonal transport after infection of the cell bodies. Prototypic strains of EV-D68 depended on sialic acid for axonal infection and transport, while contemporary circulating strains isolated during the 2014 EV-D68 outbreak did not. The pattern of infection did not correspond with the ICAM-5 distribution and expression in either human tissue, the mouse model, or the iPSC motor neurons.IMPORTANCEEnterovirus D68 (EV-D68) infections are on the rise worldwide. Since 2014, the United States has experienced biennial spikes in EV-D68-associated acute flaccid myelitis (AFM) that have left hundreds of children paralyzed. Much remains to be learned about the pathogenesis of EV-D68 in the central nervous system (CNS). Herein we investigated the mechanisms of EV-D68 CNS invasion through neuronal pathways. A better understanding of EV-D68 infection in experimental models may allow for better prevention and treatment strategies of EV-D68 CNS disease.

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