Characterization of Plaque-Sized Variants of Daniel's (DA) Strain in Theiler's Virus-Induced Epilepsy

Abstract

Epilepsy is a complex neurological disease characterized by recurrent seizures. Patients with viral encephalitis have a 16-fold increased risk of developing epilepsy, and this risk can persist for about 15 years after the occurrence of initial viral infection. Theiler's murine encephalomyelitis virus (TMEV) infection induces a well-characterized experimental model of epilepsy in C57BL/6 mice. In response to intracerebral (I.C.) injection of Daniel's (DA) strain of TMEV, there is vigorous immune response, which is detrimental to neurons and contributes to acute seizures, rendering mice susceptible to epilepsy. A comparative in vivo challenge study with either one of the two variants of the DA strain, small (DA-D_S) or large (DA-C_L) plaque forming variants, revealed differences in the diseases they induced in C57BL/6 mice. Compared to DA-C_L-, DA-D_S-infected mice exhibited significantly more seizures, higher clinical scores, neuroinflammation, and neuronal damage (mainly in the CA1-CA2 regions of hippocampus). Moreover, the brains of DA-D_S infected mice contained approximately five-fold higher virus than those of DA-C_L infected mice. A sequence comparison of the DA-C_L and DA-D_S genome sequences showed mutations in the leader (L) and L* proteins of DA-C_L variant, which may be the cause of attenuating phenotype of DA-C_L variant in the C57BL/6 mouse model of epilepsy.

Figure 1

Experimental timeline. The acute phase of disease is defined as up to day 7 p.i., during which seizures, non-epileptic clinical illness, weights, viral burden, inflammation and CNS pathology were determined. The chronic phase is defined as from week 2 to 11 p.i. or until the termination of mice. Chronic phase measures included weights, and histological analyses of the CNS.

Figure 2

Seizures induced by infection with DA-D_S were significantly more frequent and more severe than those induced by DA-C_L. To record seizures among infected groups, mice were observed twice a day for a week following infection. (a) The % of seized mice in each group was calculated as (number of seized mice/total number of infected mice) × 100 (****p < 0.0001 by Fisher’s exact test). (b) The seizure frequency was calculated as number of seizures recorded per mouse during acute phase (****p < 0.0001 by t test with Welch’s correction). The data are only from seized mice in DA-C_L and DA-D_S groups, N = 4 and 31, respectively. (c) The severity of seizures was determined based on the Racine scoring system, where score 0 indicated no seizure occurrence, and score 5 indicated tonic-clonic seizures. (d) The number of seized mice (e) and the duration of seizures were recorded each day until day 7 p.i. (*p < 0.05 by Repeated measures one-way ANOVA with post test for linear trend). Graphs (a–d) show pooled results from four separate experiments expressed as number/percent/mean ± SEM, N = 33 per infected group. Graph (e) shows pooled results from three separate experiments expressed as mean ± SEM, the data are from seized mice in DA-C_L and DA-D_S groups, N = 3 and 22, respectively.

Figure 3

Mice infected with DA-D_S plaque variant displayed severe non-epileptic clinical signs and weight loss. (a) Mice were observed daily as described in Materials and Methods for signs of non-epileptic clinical disease during the acute phase (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Mann-Whitney test). Mice from the control and infected groups were weighed (b) daily until day 7 p.i. and then (c) once weekly until week 5 p.i. (*p < 0.05, **/^##p < 0.01, ***/^###p < 0.001, ****/^####p < 0.0001 by Repeated Measures two-way ANOVA with Tukey’s multiple comparisons test). Graphs (a,b) show pooled results from four separate experiments expressed as mean ± SEM, N = 4 in control group and N = 32–33 per infected group. Graph (c) shows pooled results from two separate experiments expressed as mean ± SEM, N = 4 in control group and N = 13 per infected group.

Figure 4

Increased viral burden in mice infected with DA-D_S plaque-variant. (a) Using plaque assay, viral burden was determined in brain and spinal cord homogenates from day 7 p.i. (b) Viral-antigen positive cells were detected in the hippocampus using rabbit anti-TMEV at day 7 p.i. The number of viral-antigen positive cells in the left and right hippocampi was enumerated and compared between the infected groups. (**p < 0.01, ***p < 0.001 by t test with Welch’s correction). (c,d) The ⇦ indicates viral-antigen positive cells in the CA1 and CA2 pyramidal layers of the hippocampus. Some of the cortical neurons surrounding the CA1 and CA2 pyramidal layers also stained positive for viral-antigen. Graph (a) shows mean ± SEM, N = 10 in DA-C_L group and N = 11 in DA-D_S group. Graph (b) shows mean ± SEM, N = 4/infected group. Cornu ammonis1 (CA1), cornu ammonis3 (CA3), and dentate gyrus (DG).

Figure 5

Increased glial pathology among DA-D_S infected mice. Microglia/macrophages were detected with an antibody against Iba1, astrocytes with an antibody against Gfap and counter stained with Hoechst. (a) Schematic diagram of the coronal section of hippocampus depicting different areas of CA1 field. (b,c) Images of corresponding regions of hippocampus from control group depicting Iba1+ cells, and (h,i) images depicting Gfap+ cells. In comparison to control, (d–g) activated macrophage/microglia and (j–m) astrocytes were found in CA1 region of hippocampus in both DA-C_L- and DA-D_S-infected groups. The ⇦ indicates Iba1+ or Gfap+ cells lining PVCs, and the Δ indicates activated cells. Cornu ammonis1 (CA1), cornu ammonis3 (CA3), dentate gyrus (DG), stratum lacunosum-moleculare (SLM), and peri-vascular cuffs (PVCs).

Figure 6

Extensive inflammation and neuronal damage in the hippocampus of mice infected with DA-D_S variant. (a) H&E stained coronal section of hippocampus from control mice. Hippocampal regions from the infected groups were analyzed at days 3 (b,f) and 7 (c,g) and weeks 5 (d,h) and 11 (e,i) p.i. for histopathology. (b–e) are the representative images from DA-C_L-infected group and (f–i) are the representative images from DA-D_S-infected group. At day 7 p.i., the percent loss of CA1-CA2 neurons was estimated and ranked on a scale of 0 to 10. (c) A representative section of hippocampus from DA-C_L-infected group presenting CA1-CA2 damage score 2. (g) A representative section of hippocampus from DA-D_S-infected group presenting CA1-CA2 damage score 10. (j) The damage to the CA1 and CA2 pyramidal layers (**p < 0.01 by Mann-Whitney test) and (k) the intensity of inflammation as determined by the number of perivascular cuffs and inflammatory foci (*p < 0.05 by t test with Welch’s correction) were compared between the infected groups at day 7 p.i. The ▼ indicates neuronal loss and the *indicates inflammation in the images. Graphs show mean ± SEM, N = 5 per infected group. Cornu ammonis1 (CA1), cornu ammonis2 (CA2), cornu ammonis3 (CA3), dentate gyrus (DG), alveus (Av.), hippocampal fissure (HF) and stratum lacunosum-moleculare (SLM).

Figure 7

Increased immune cell infiltration into the CNS of mice following DA-D_S-infection. As described in Materials and Methods, brain leukocytes from control and infected mice were collected at day 3 p.i. using Percoll gradient, stained for cell specific markers, and analyzed by flow cytometry. (a) The total number of brain leukocytes collected. (b) % CD45.2int CD11b+, % CD45.2hi CD11b+, and % CD45.2+ Gr1+ cells of the total brain leukocytes. Cells were gated based upon their CD45.2int/low CD11b+ or CD45.2+ Gr1+ expression. Graphs show pooled results from four separate experiments expressed as mean ± SEM, N = 5 in control group and N = 13 per infected group. (*p < 0.05, **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons test). Intermediate (int) and high (hi).