Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells
Brown DM, Hixon AM, Oldfield LM, Zhang Y, Novotny M, Wang W, Das SR, Shabman RS, Tyler KL, Scheuermann RH
Enterovirus D68 (EV-D68) has historically been associated with respiratory illnesses. However, in the summers of 2014 and 2016, EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. To assess the potential neurotropism of EV-D68, we utilized the neuroblastoma-derived neuronal cell line SH-SY5Y as a cell culture model to determine if differential infection is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y were assessed using multiple assays: virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral binding and entry were at least partly responsible for the differential infectivity phenotype. Transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains that could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help identify the signatures of virulence to confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains. Since the EV-D68 outbreak during the summer of 2014, evidence of a causal link to a type of limb paralysis (AFM) has been mounting. In this article, we describe a neuronal cell culture model (SH-SY5Y cells) in which a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism. We confirmed the difference in neurotropism using primary human neuron cell cultures and with a mouse paralysis model. Using the SH-SY5Y cell model, we determined that a barrier to viral entry is at least partly responsible for neurotropism. SH-SY5Y cells may be useful in determining if specific EV-D68 genetic determinants are associated with neuropathogenesis, and replication in this cell line could be used as rapid screening tool for identification of neurotropic EV-D68 strains. This may assist with better understanding of pathogenesis and epidemiology and with the development of potential therapies.