Article Figures & Data
Figures
- Figure 1 Diagram Illustrating the Patient Cohorts and the Experimental Workflow With Their Serum Samples
The numbers of patients' serum samples collected in the different categories of ZIKV-GBS (patients with ZIKV who developed GBS), ZIKV-OND (patients with ZIKV who displayed other neurologic diseases), ZIKV-CON (patients with ZIKV who never developed clinical neurologic disease), DENV-CON (patients with DENV who never developed clinical neurologic disease), ALS (patients with amyotrophic lateral sclerosis, a progressive nervous system disease that affects nerve cells in the brain and spinal cord, causing loss of muscle control), dysferlin (patients with dysferlinopathy, a muscle disease that has a slow progression of muscle weakness and atrophy), and healthy controls (n, number of patient samples). The patients' serum samples were initially screened for their autoantibody reactions against different neuronal cell culture types and from which the sera from patient A to D's serum samples were then tested for their reactions in additional assays.
- Figure 2 Complement-Dependent Demyelinating Activity of ZIKV Patient Sera
The autoantibodies in the sera of ZIKV-CON patient B (BQ008) reacted with myelin internodes and fixed complement (C3c deposition; green) over myelin internodes (MBP; red) when complement was present from 20% normal human sera (NHS) (A) but not when only NHS was added to the cells (B). Counterstaining for NF200 (blue, axons) and MBP (red) is also shown. (C) Fluoromyelin labeling (red) from live imaging of a myelinated coculture before (above) and 16 hours after (below) incubation with patient B's serum at a 1:50 dilution in the presence of complement in 20% NHS, while (D) shows the results of time-lapse stills of the receding myelin internode junctions shown from right to left over time (arrows). (E) shows the overlaid example images of live myelin staining at baseline (red) and after 24 hours of incubation (silver) with patient B's serum with or without 20% NHS containing complement. (E.b) shows numerous myelin internodes are either lost completely or extensively fragmented after 24 hours (shown by arrows), while (E.a) without NHS containing complement the distribution of the myelin was essentially unchanged over 24 hours. (F) The mean internodal length was significantly reduced at 24 hours compared with baseline in the ZIKV-CON + NHS group only (****p < 000.1, 2-way ANOVA with Sidak correction); ns = not significant. MBP = myelin basic protein; ZIKV = Zika virus; ZIKV-CON = ZIKV-infected patients without neurologic complications.
- Figure 3 Identification of a ZIKV Patient's Autoantibodies Against Myelin Antigen: Immunoprecipitation and Peptide Mass Spectrometry
Immunofluorescent photomicrographs of (A) a positive control patient's serum with autoantibodies against the known nerve antigen NF155 and (B) ZIKV-CON patient B's serum autoantibody reactions against unknown antigens enriched in abaxonal myelin, with higher magnifications of the nodal regions shown as insets (far right). Scale bars, 20 μm. Counterstaining for NF200 (blue) and MBP (red) is also shown. (C) Proteins identified in immunoprecipitates from ZIKV-CON patient B serum and NF155+ serum-treated coculture lysates. The t test of enriched proteins presented as a volcano plot. Significantly enriched membrane proteins are shown as red squares. NFASC protein isoforms from a human and rat database are grouped together (blue circle). AHNAK2 = desmoyokin; CD44 = P-glycoprotein 1; ITGA6 = integrin alpha 6. MBP = myelin basic protein; NEP = neprilysin; SLC12A2 = solute carrier 12/Na-K-2Cl cotransporter A2; TGBR3 = transforming growth factor receptor 3; ZIKV = Zika virus; ZIKV-CON = ZIKV-infected patients without neurologic complications.
- Figure 4 Removal of ZIKV-CON Patient B's Serum IgG Autoantibody Target Antigen(s) in Myelinated Sensory Neuron Cultures by Lipid Extraction
(A) Immunofluorescent photomicrographs of ZIKV-CON patient B's serum IgG autoantibodies showing strong staining with untreated (control) abaxonal membranes of all myelinating SCs, which was particularly intense at the Schwann cell microvilli overlying the node of Ranvier (arrowhead) while (B) weakly staining these same cells after solvent treatment (solvent). The control reactions for neuronal (NF200) and myelin (MBP) markers remained unchanged. For this study, the serum-treated fixed cultures were incubated with a mixture of chloroform, methanol, and water at a ratio of 4:8:3 for 1 hour on ice to remove glycolipids. The white boxes indicate regions of fluorescence profiling per internode (5 × 20 μm). Arrows indicate weak paranode-like IgG antibody binding after lipid extraction. (C) Fluorescence intensity profiles of the human IgG autoantibodies and MBP immunolabeling across the internodes of control and solvent-treated cultures. Data are presented as mean (± SEM in gray) of 2 internodes per field of view, 12–14 fields of view from 2 independent experiments. (D) Quantification of IgG:MBP fluorescence ratio. Student unpaired t test, p < 0.0001, t = 5.053, n = 24–28 internode profiles per group. MBP = myelin basic protein; ZIKV = Zika virus; ZIKV-CON = ZIKV-infected patients without neurologic complications.
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