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

mBio. 2019 Oct 22;10(5):e02370-19. doi: 10.1128/mBio.02370-19.


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.

Keywords: acute flaccid myelitis; astrocyte; enterovirus; neuron; neurotropism; picornavirus.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Astrocytes / metabolism
  • Astrocytes / virology
  • Brain / metabolism
  • Brain / virology
  • Enterovirus D, Human / metabolism*
  • Enterovirus D, Human / pathogenicity*
  • Female
  • In Vitro Techniques
  • Male
  • Mice
  • N-Acetylneuraminic Acid / metabolism*
  • Neuraminidase / metabolism
  • Neurons / metabolism
  • Neurons / virology


  • Neuraminidase
  • N-Acetylneuraminic Acid