Understanding Enterovirus D68-Induced Neurologic Disease: A Basic Science Review

Abstract

In 2014, the United States (US) experienced an unprecedented epidemic of enterovirus D68 (EV-D68)-induced respiratory disease that was temporally associated with the emergence of acute flaccid myelitis (AFM), a paralytic disease occurring predominantly in children, that has a striking resemblance to poliomyelitis. Although a definitive causal link between EV-D68 infection and AFM has not been unequivocally established, rapidly accumulating clinical, immunological, and epidemiological evidence points to EV-D68 as the major causative agent of recent seasonal childhood AFM outbreaks in the US. This review summarizes evidence, gained from in vivo and in vitro models of EV-D68-induced disease, which demonstrates that contemporary EV-D68 strains isolated during and since the 2014 outbreak differ from historical EV-D68 in several factors influencing neurovirulence, including their genomic sequence, their receptor utilization, their ability to infect neurons, and their neuropathogenicity in mice. These findings provide biological plausibility that EV-D68 is a causal agent of AFM and provide important experimental models for studies of pathogenesis and treatment that are likely to be difficult or impossible in humans.

Keywords: acute flaccid myelitis; enterovirus D68; experimental models; mouse models; neuropathogenesis; paralysis.

Figure 1

Contemporary strains of enterovirus D69 (EV-D68) cause paralysis in neonatal mice. (A) Examples of limb paralysis (arrows) in 3 different neonatal Swiss Webster (SW) mice following intracerebral (IC) injection of the contemporary EV-D68 strain MO/14-18947. Images are of mice at day post infection (dpi) 3, 12, and 28. (B) A cervical spinal cord section of a mouse at 100× original magnification following IC injection with EV-D68 MO/14-18947 that developed right forelimb paralysis on day 4 post-injection. Loss of motor neurons (green, labeled with choline acetyltransferase/ChAT) is observed in the right (“R side”) anterior horn, corresponding to the affected side. (C) 200× and (D) 600× images from a left anterior horn in an IC-injected mouse at 3 dpi before the onset of paralysis showing EV-D68 antigen in an intact cluster of motor neurons. The box in (C) represents the area imaged at 600× in (D). For all images, neurons (magenta) are labelled with NeuN, a general neuron marker, and nuclei (blue) are labeled with Hoechst 33342. Scale bars for 100× original magnification are 400 μm, 200× are 200 μm, 600× are 50 μm. (E) Tissue viral loads of 15296-virus (an EV-D68 strain produced by reverse engineering) in infected neonatal BALB/c mice. Samples of blood, brain, heart, intestine, kidney, liver, lung, muscle, spinal cord, and spleen were collected from infected mice following i.p. challenge with 5 LD50 of 15296-virus at 1, 3, 5 and 7 dpi (n = 3 per time point) or control mice given Dulbecco’s modified eagle medium (DMEM) at 0 days post-infection. Viral load was measured by real-time PCR, and the results represent the mean viral load ± SD. (F) Age dependence of EV-D68-induced disease and mortality. ICR mice (n = 11–15/group were intraperitoneally (IP) injected with 2.0 × 10⁶ TCID50 of US/MO/14-18947 per mouse at 1, 5, 7, 9, or 12 days of age. Survival and clinical score were then monitored and recorded daily. Clinical scores were graded as follows: 0, healthy; 1, lethargy and reduced mobility; 2, limb weakness; 3, limb paralysis; 4, death. Images taken from Hixon et al., 2017 (A–D), Sun et al., 2019 (E), and Zhang et al., 2018 (F).