Evaluating Treatment Efficacy in a Mouse Model of Enterovirus D68-Associated Paralytic Myelitis

Abstract

Background: Enterovirus D68 (EV-D68)-associated acute flaccid myelitis (AFM) is a devastating neurological disease for which there are no treatments of proven efficacy. The unpredictable temporal and geographic distribution of cases and the rarity of the disease make it unlikely that data from randomized controlled trials will be available to guide therapeutic decisions. We evaluated the following 3 widely used empirical therapies for the ability to reduce the severity of paralysis in a mouse model of EV-D68 infection: (1) human intravenous immunoglobulin (hIVIG), (2) fluoxetine, and (3) dexamethasone.

Methods: Neonatal mice were injected intramuscularly with a human 2014 EV-D68 isolate that reliably induces paralysis in mice due to infection and loss of spinal cord motor neurons. Mice receiving treatments were evaluated for motor impairment, mortality, and spinal cord viral load.

Results: hIVIG, which contained neutralizing antibodies to EV-D68, reduced paralysis in infected mice and decreased spinal cord viral loads. Fluoxetine had no effect on motor impairment or viral loads. Dexamethasone treatment worsened motor impairment, increased mortality, and increased viral loads.

Conclusion: Results in this model of EV-D68-associated AFM provide a rational basis for selecting empirical therapy in humans and establish this model as a useful system for evaluating other potential therapies.

Keywords: Enterovirus D68; acute flaccid myelitis; mouse model; paralysis; therapies.

Figure 1.

Intramuscular enterovirus D68 (EV-D68) injection model for tracking paralytic disease over time. A, Dose response curve for mice given 10 (n = 21), 10² (n = 19), 10³ (n = 27), and 10⁴ (n = 14) 50% tissue culture infectious doses (TCID₅₀) of EV-D68 IL/14-18952 by intramuscular inoculation, as quantified by motor impairment score for mice surviving to the experimental end point 21 days after infection. B, Viral growth in muscle and spinal cord tissue specimens following intramuscular injection with 10³ TCID₅₀ of EV-D68 IL/14-18952 (n = 10 per time point). Error bars are standard errors of the mean.

Figure 2.

Enterovirus D68 (EV-D68) neutralizing antibody titer in human intravenous immunoglobulin (hIVIG) versus immune mouse serum. A, Neutralizing antibody titers of hIVIG used for in vivo experiments against select EV-D68 strains, including the ancestral EV-D68 strain Fermon and three 2014 strains of EV-D68 from different clades: MO/14-18947 (clade B1), IL/14-19852 (clade B), and KY/14-18953 (clade A). B, Neutralizing antibody titers in immune serum from mice inoculated with intramuscular EV-D68 IL/14-18952 against the infecting strain IL/14-18952, the closely related strain MO/14-18947, and the more distantly related KY/14-18953 and Fermon strains. Boxes represent 95% confidence intervals with a line at the median. LLD, lower limit of detection; ULD, upper limit of detection.

Figure 3.

Human intravenous immunoglobulin (hIVIG) reduces enterovirus D68 (EV-D68)–induced motor impairment and viral loads. A, Motor impairment scores for mice infected by intramuscular inoculation with 10³ EV-D68 IL/14-18952 and then given hIVIG by intraperitoneal injection at either 1 day (n = 8), 3 days (n = 11), 4 days (n = 11), or 6 days (n = 12) after infection. Control mice received an intraperitoneal injection of phosphate-buffered saline (n = 40). Data for animals surviving to the end of the study were graphed. B and C, Mice were treated with hIVIG (n = 10) or control (n = 7) on day 3 after infection. On day 6 after infection, muscle (open squares) and spinal cord (filled circles) tissue specimens were collected for viral titer analysis. Error bars are standard errors of the mean. *P ≤ .05  , **P ≤ .01, and ****P ≤ .0001.

Figure 4.

Fluoxetine has no effect on enterovirus D68 (EV-D68)–induced motor impairment and viral loads. A, Motor impairment scores for mice infected by intramuscular injection with 10³ 50% tissue culture infectious doses (TCID₅₀) of EV-D68 IL/14-18952 and then given fluoxetine by intraperitoneal injection for 7 days on days 0–6 after infection at 3 different concentrations (0.75 mg/kg [n = 7], 1.5 mg/kg [n = 9], or 3.0 mg/kg [n = 7]) versus vehicle control (n = 29). Treatment started the same day as viral infection, defined as day 0 after infection, and ended on day 6 after infection. Data for animals surviving to the end of the study were graphed. B and C, Mice were treated with 3.0 mg/kg fluoxetine (n = 11) for 7 days (days 0–6 after infection) or vehicle (n = 11) following intramuscular injection of 10³ TCID₅₀ EV-D68 IL/14-18952. On day 6 after infection, several hours after the final dose of fluoxetine, muscle (open squares) and spinal cord (filled circles) tissue specimens were collected for viral titer analysis. Error bars are standard errors of the mean. NS, not significant.

Figure 5.

Dexamethasone (Dex) significantly worsens enterovirus D68 (EV-D68)–induced motor impairment and increases viral loads. A, Survival curves for mice given intraperitoneal Dex either 3 and 4 days (early treatment; n = 12) or 6 and 7 days (late treatment; n = 12) following intramuscular infection with 10³ TCID₅₀ EV-D68 IL/14-18952 versus controls receiving virus with vehicle (solid black line; n = 12) or Dex only (dashed gray line; n = 21). B, Motor impairment scores of mice that died who received treatment with early (squares) and late (triangles) Dex treatment versus the average motor impairment score of surviving infected, vehicle treated controls (filled diamonds). C and D, Mice were given intramuscular 10³ TCID₅₀ EV-D68 IL/14-18952 followed by Dex on days 3 and 4 after infection (n = 20) or vehicle (n = 20). On day 6 after infection, muscle (open squares) and spinal cord (filled circles) tissue specimens were collected from surviving mice for viral titer analysis. *P ≤ .05, **P ≤ .01, and ***P ≤ .001. NS, not significant.