Inflammatory Biomarkers Associated with Lethal Rift Valley Fever Encephalitis in the Lewis Rat Model

Abstract

Rift Valley fever (RVF) is an emerging viral disease that causes significant human and veterinary illness in Africa and the Arabian Peninsula. Encephalitis is one of the severe complications arising from RVF virus (RVFV) infection of people, and the pathogenesis of this form of RVF is completely unknown. We use a novel reproducible encephalitic disease model in rats to identify biomarkers of lethal infection. Lewis rats were infected with RVFV strain ZH501 by aerosol exposure, then sacrificed daily to determine the course of infection and evaluation of clinical, virological, and immunological parameters. Weight loss, fever, and clinical signs occurred during the last 1-2 days prior to death. Prior to onset of clinical indications of disease, rats displayed marked granulocytosis and thrombocytopenia. In addition, high levels of inflammatory chemokines (MCP-1, MCS-F, Gro/KC, RANTES, and IL-1β) were detected first in serum (3-5 dpi) followed by brain (5-7 dpi). The results of this study are consistent with clinical data from human RVF patients and validate Lewis rats as an appropriate small animal model for RVF encephalitis. The biomarkers we identified here will be useful in future studies evaluating the efficacy of novel vaccines and therapeutics.

Keywords: Rift Valley fever virus; aerosol exposure; neurological disease; respiratory infection; viral encephalitis.

FIGURE 1

Weight loss and temperature changes in Rift Valley fever virus (RVFV)-infected Lewis rats. Changes in (A) body weight, (B) temperature over the duration of the experiment. The gray shaded box on both graphs represents the window of clinical disease (5–7 dpi).

FIGURE 2

Virus dissemination over time. Virus was measured in the indicated tissue by plaque assay (solid squares; pfu/g tissue) or taqman RT-PCR (open circles; pfu equivalents/g tissue). Horizontal line on each graph represents the limit of detection of the plaque assay (50 pfu). The gray shaded box on all graphs represents the window of clinical disease (5–7 dpi). For (A–C,F), the M on the x-axis stands for ‘moribund’ and represents historical samples from previous Lewis rat experiments (correspond to 7–8 dpi). Tissue samples were taken at the time the rat was moribund and are included here for comparison purposes. These samples are not available for (D) and (E).

FIGURE 3

Histologic images of the brain of RVFV-infected Lewis Rats. (A) H&E; Rat R819 taken 7 dpi; low magnification showing lymphocytic meningitis with apoptosis of the subcortical neurons. (B) IHC; Rat R819 taken 7 dpi; RVFV Ag+ cells (reddish/brown staining) in the cortex with lymphocytic meningitis. (C) IHC; Rat R818 taken 7 dpi; low magnification illustrating the large number of RVFV Ag+ cells in the cortex with marked lymphocytic meningitis. (D) H&E; Rat R819 taken 7 dpi; high magnification of subcortical cells with characteristic appearance of apoptosis (arrows). (E) H&E; Rat R821 taken 6 dpi; subcortical cells with characteristic appearance of apoptosis (arrows). (F) IHC; Rat R818 taken 7 dpi; high magnification of RVFV Ag+ cells in cortex.

FIGURE 4

Vasculitis in the brain of RVFV-infected Lewis rats. (A) H&E; Rat R821 taken 6 dpi; vasculitis consisting of lymphocytes (thin arrows), area of concentrated histiocytes (thick arrow), and area of concentrated neutrophils (arrowhead). (B) H&E; Rat R818 taken 7 dpi; vasculitis with lymphocytes, histiocytes, and neutrophils. Large number of neutrophils are seen in the bottom half of the photo.

FIGURE 5

Apoptotic morphology of cortical neurons. Rat R818 taken 7 dpi. (A) H&E; cerebral cortex with morphologically apoptotic cells (asterisks). (B) IHC; RVFV Ag+ neurons in the cerebral cortex, two with apoptotic appearance (asterisks).

FIGURE 6

Changes in blood cells over the course of infection. The arrow represents a significant decrease in blood lymphocytes at that time point, as measured by t-test. The black box represents the time point at which both granulocytes and platelets differ significantly from controls (as measured by ANOVA with multiple comparison test), and these differences persist through the end of infection. The gray shaded box represents the window of clinical disease (5–7 dpi).

FIGURE 7

Liver metabolic enzymes during the course of RVFV infection of Lewis rats. 14-parameter blood chemistry analysis was performed on each rat at the time of sacrifice. Alanine transaminase (ALT; a marker of liver cell damage) and alkaline phosphatase (ALP; produced by liver cells) are shown. Significance was evaluated using one-way ANOVA and no significant differences over time were found (p = 0.1 and p = 0.08 for ALT and ALP, respectively).

FIGURE 8

Cytokine and chemokine dysregulation in serum and brain of RVFV-infected Lewis rats. Serum and homogenized brain tissue were analyzed using the Bio-Plex Pro Rat Cytokine 23-plex assay. All analytes were tested for significance using one-way ANOVA. The five cytokines shown in this figure were statistically significant for both serum (left column) and brain (right column). The level of significance by ANOVA is reflected by the number of asterisks on each graph title, where ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001. Individual time points were compared to uninfected controls by t-test. Significant time points are indicated by the black box. The gray shaded box on all graphs represents the window of clinical disease (5–7 dpi).

FIGURE 9

Cytokine and chemokine dysregulation in the brain of RVFV-infected Lewis rats. The eight cytokines shown in this figure were statistically significant in brain but not serum. The level of significance by ANOVA is reflected by the number of asterisks on each graph title, where ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001. Individual time points were compared to uninfected controls by t-test. Significant time points are indicated by the black box. The gray shaded box on all graphs represents the window of clinical disease (5–7 dpi).

FIGURE 10

Course of neurological disease in RVFV-infected Lewis rats. Timing of clinical and virological events after aerosol exposure to RVFV. The clinical window is from 5 dpi until death of the rat, and consists of behavioral and appearance changes, weight loss, and fever. Rats succumb to disease between 6 and 8 dpi. Cytokine/chemokine dysregulation in the blood starts as early as 1 dpi (RANTES) and extends to 5 dpi (MCP-1, M-CSF, and Gro/KC). Changes in CBC start at 4 dpi and persist throughout infection. Live virus is found in the brain from 5 dpi onwards, along with significant pathology and cytokine/chemokine dysregulation. Virus-specific antibody responses are not apparent until 6 dpi.