The severity of murray valley encephalitis in mice is linked to neutrophil infiltration and inducible nitric oxide synthase activity in the central nervous system

Abstract

A study of immunopathology in the central nervous system (CNS) during infection with a virulent strain of Murray Valley encephalitis virus (MVE) in weanling Swiss mice following peripheral inoculation is presented. It has previously been shown that virus enters the murine CNS 4 days after peripheral inoculation, spreads to the anterior olfactory nucleus, the pyriform cortex, and the hippocampal formation at 5 days postinfection (p.i.), and then spreads throughout the cerebral cortex, caudate putamen, thalamus, and brain stem between 6 and 9 days p.i. (P. C. McMinn, L. Dalgarno, and R. C. Weir, Virology 220:414-423, 1996). Here we show that the encephalitis which develops in MVE-infected mice from 5 days p.i. is associated with the development of a neutrophil inflammatory response in perivascular regions and in the CNS parenchyma. Infiltration of neutrophils into the CNS was preceded by increased expression of tumor necrosis factor alpha and the neutrophil-attracting chemokine N51/KC within the CNS. Depletion of neutrophils with a cytotoxic monoclonal antibody (RB6-8C5) resulted in prolonged survival and decreased mortality in MVE-infected mice. In addition, neutrophil infiltration and disease onset correlated with expression of the enzyme-inducible nitric oxide synthase (iNOS) within the CNS. Inhibition of iNOS by aminoguanidine resulted in prolonged survival and decreased mortality in MVE-infected mice. This study provides strong support for the hypothesis that Murray Valley encephalitis is primarily an immunopathological disease.

FIG. 1

(A) Cumulative mortality profile of 21-day-old Swiss mice infected with MVE BH3479. Groups of 40 mice of either sex were inoculated i.p. with 10⁴ PFU of BH3479 and were observed for 14 days of signs of illness and death. The percents cumulative mortality at the indicated time points are shown. No mock-infected mice died during the experiment. (B) Growth of MVE BH3479 in the brains of 21-day-old Swiss mice after peripheral inoculation. Groups of 50 mice of either sex were inoculated i.p. with 10⁴ PFU of virus. At the indicated time points, mice were terminally anesthetized and the brains were removed and prepared as 10% suspensions in Hanks’ balanced salt solution. Infectivity titers within infected brains were determined by plaque formation on Vero cell monolayers. Infectivity titers are expressed as log₁₀ PFU/g of tissue. Each time point represents the average of four mice; titers at each time point did not vary more than fourfold.

FIG. 2

Localization of MVE BH3479 RNA by ISH and of apoptotic cells by in situ TUNEL assay in the brains of Swiss mice. The ISH probe was DIG-labelled MVE E gene RNA of negative polarity; the hybridized probe was detected with anti-DIG alkaline phosphatase and fast red to produce a red cytoplasmic stain. TUNEL positivity was detected with biotinylated dUTP, streptavidin horseradish peroxidase, and diaminobenzidine to produce a brown nuclear stain. Frozen tissue sections (15 μm) were cut on a cryostat and counterstained with hematoxylin. (A) Coronal section through the anterior olfactory nucleus at 6 days p.i. A double-labelled neuron (long arrow) is seen in the center of the picture. Numerous MVE-infected neurons with TUNEL-negative nuclei are present (short arrows). Bar, 50 μm. (B) Coronal section through the hippocampal formation at 6 days p.i. A double-labelled neuron (long arrow) is seen within a heavily infected region of CA3 (indicated by the no. 3). A TUNEL-positive inflammatory cell is seen adjacent to the double-labelled neuron (short arrow). Bar, 50 μm.

FIG. 3

Identification of infiltrating neutrophils in the brains of MVE BH3479-infected Swiss mice. Neutrophils were identified by using the rat anti-mouse neutrophil MAb RB6-8C5. Bound antibody was detected by using modified Hanker-Yates substrate (13, 32) to produce an opaque black deposit. Frozen tissue sections (8 μm) were cut on a cryostat and counterstained with hematoxylin. (A) Coronal section through the cerebral cortex at 6 days p.i. Many neutrophils can be seen adhering to neurons (neuronophagia; arrows). Bar, 80 μm. (B) Coronal section through the hippocampal formation at 7 days p.i. Foci of neutrophils (arrows) can be seen adhering to neurons within the CA3 (indicated by the no. 3) region. Bar, 50 μm.

FIG. 4

Representative RT-PCR and Southern blot analysis of GAPDH, TNF-α, N51/KC, and iNOS mRNA from MVE-infected mouse brains at 2 to 8 days after i.p. inoculation of 10⁴ PFU of BH3479 (two to four mice per time point); individual mouse brain samples are marked (lanes a through d) at each time point. Negative controls consisted of distilled water and mock-infected mouse brain RNA during the RT-PCR; mock-infected mouse brain samples are labelled (lanes m) at each time point. Predicted sizes of the RT-PCR products are as follows: 520 bp for GAPDH, 427 bp for the MVE E gene, 374 bp for TNF-α, 539 bp for N51/KC, and 492 bp for iNOS. Bands of the appropriate size were amplified in each case, indicating that the PCR products had been amplified from mRNA templates and not from contaminating genomic DNA.