Infection and injury of neurons by West Nile encephalitis virus

Abstract

West Nile virus (WNV) infects neurons and leads to encephalitis, paralysis, and death in humans, animals, and birds. We investigated the mechanism by which neuronal injury occurs after WNV infection. Neurons in the anterior horn of the spinal cords of paralyzed mice exhibited a high degree of WNV infection, leukocyte infiltration, and degeneration. Because it was difficult to distinguish whether neuronal injury was caused by viral infection or by the immune system response, a novel tissue culture model for WNV infection was established in neurons derived from embryonic stem (ES) cells. Undifferentiated ES cells were relatively resistant to WNV infection. After differentiation, ES cells expressed neural antigens, acquired a neuronal phenotype, and became permissive for WNV infection. Within 48 h of exposure to an exceedingly low multiplicity of infection (5 x 10(-4)), 50% of ES cell-derived neurons became infected, producing nearly 10(7) PFU of infectious virus per ml, and began to die by an apoptotic mechanism. The establishment of a tractable virus infection model in ES cell-derived neurons facilitates the study of the molecular basis of neurotropism and the mechanisms of viral and immune-mediated neuronal injury after infection by WNV or other neurotropic pathogens.

FIG. 1.

Histopathology, antigen expression, and leukocyte infiltration of WNV infection. (A to C) Histopathology of WNV infection in C57BL/6 mice. Spinal cords from mice that were uninfected (A), infected with WNV but not paralyzed (B), or infected and paralyzed (C) were harvested at 10 days, sectioned, and stained with hematoxylin and eosin (H&E). Typical sections are shown after reviewing >10 independent spinal cords from uninfected, infected and nonparalyzed, or infected and paralyzed wild-type mice. Thick black arrows identify infiltrating leukocytes and thin black arrows denote degenerating neurons. (D to F) WNV antigen expression in the spinal cords of uninfected (D), WNV infected and not paralyzed (E), or infected and paralyzed (F) mice. Spinal cords were harvested 10 days after infection, sectioned, and stained with a rat anti-WNV polyclonal serum. Typical sections are shown after reviewing between 5 and 10 independent spinal cords and brains from uninfected, infected and nonparalyzed, or infected and paralyzed wild-type mice. Green arrows indicate heavily infected neurons. (G to I) Leukocyte infiltration in the spinal cord. Sections from uninfected (G), WNV infected and not paralyzed (H), and infected and paralyzed (I) mice were stained for infiltrating leukocytes with a monoclonal antibody against CD45, a common leukocyte antigen. Red arrows indicate CD45⁺ leukocytes.

FIG. 2.

WNV infection in undifferentiated ES cells. Undifferentiated ES 129 (A) or C57BL/6J (B) cells were infected with increasing amounts of WNV. At 48 h postinfection, cells and supernatants were harvested. Viral plaque assays were performed in BHK21 cells to determine the viral titers, which are expressed in PFU per milliliter. The percentage of cells infected was determined by a flow cytometric assay that reliably detects ≥1% of infected cells by intracellular staining of the viral envelope protein.

FIG. 3.

WNV infection of differentiated ES cells. (A) Flow cytometric scatter and fluorescence dot plots of WNV-infected ESNC. Differentiation with retinoic acid yielded two populations of neurons by forward scatter (FSC) and side scatter (SSC) analysis (top). The larger population expressed higher levels of neurofilament (NF) and WNV antigens (compare middle and bottom panels) after infection. (B) Dose-dependent infection of ESNC. ESNC from 129/Sv (top) or C57BL/6 (bottom) mice were infected with increasing amounts of WNV and harvested 2 days later for flow cytometric and viral plaque assays. For flow cytometric analysis, neuronal populations were separated by their relative expression (low or high) of neurofilament protein. Data are the averages of three independent experiments and error bars reflect the standard deviations. (C) Kinetics of WNV infection in ESNC. Neurons were infected at an MOI of 0.05 with WNV. At the indicated times after infection, cells were harvested for flow cytometric analysis of viral antigen and supernatants were assessed for infectious virus as described for Fig. 2. Data are the averages of three independent experiments and error bars reflect the standard deviations.

FIG. 4.

Apoptosis assays with WNV-infected neurons. (A) Flow cytometry profiles with annexin V and 7-AAD staining of uninfected (left) and WNV-infected neurons at 24 and 48 h (middle and right, respectively). (B) Summary of apoptosis data in neurons derived from ES cells. Data are the averages of three independent experiments and error bars reflect the standard deviations. (C) Intracellular staining of NS1 antigen confirms that ∼90% of neurons are infected at 48 h after infection. (D) WNV infection and neuronal phenotype in annexin V-positive apoptotic cells. Left panel, negative control (anti-DV3 antibody) staining; middle, the majority of annexin V-positive cells also express WNV NS1 protein on their surfaces; right, neurofilament protein staining on the surfaces of annexin V-positive cells. (E) DNA fragmentation in WNV-infected ESNC. ESNC were not infected or infected with WNV at an MOI of 10 and incubated for 48 h. DNA was extracted from cells and resolved by agarose gel electrophoresis. The gel mobility standards (S) are included to the right of the figure. Three independent samples are shown for each condition.

FIG. 5.

Electron micrographs of WNV-infected ESNC. Uninfected (A) or infected (B to D) (48-h time point; MOI of 10) cells were harvested, fixed, sectioned, and processed by electron microscopy. Typical sections are shown after a review of >15 independent images. WNV-infected ESNC show evidence of apoptosis, including chromatin condensation and marginalization (CC) along the nuclear membrane. Scale bar, 1 μm.

FIG. 6.

In situ TUNEL staining of spinal cord neurons after WNV infection. Sections from WNV-infected and nonparalyzed (A and B) and WNV-infected and paralyzed (C and D) mice were analyzed for cell death by TUNEL staining (A to D) and expression of the NeuN neuronal antigen (B and D). Cells that are undergoing cell death (black arrows) appear red after incubation with the chromogenic substrate. In the paralyzed mice, many of the TUNEL-positive cells stained positively for the NeuN neuronal nuclear antigen (green arrows) (D).