Characterization and pathogenesis of aerosolized eastern equine encephalitis in the common marmoset (Callithrix jacchus)

Abstract

Background: Licensed antiviral therapeutics and vaccines to protect against eastern equine encephalitis virus (EEEV) in humans currently do not exist. Animal models that faithfully recapitulate the clinical characteristics of human EEEV encephalitic disease, including fever, drowsiness, anorexia, and neurological signs such as seizures, are needed to satisfy requirements of the Food and Drug Administration (FDA) for clinical product licensing under the Animal Rule.

Methods: In an effort to meet this requirement, we estimated the median lethal dose and described the pathogenesis of aerosolized EEEV in the common marmoset (Callithrix jacchus). Five marmosets were exposed to aerosolized EEEV FL93-939 in doses ranging from 2.4 × 10¹ PFU to 7.95 × 10⁵ PFU.

Results: The median lethal dose was estimated to be 2.05 × 10² PFU. Lethality was observed as early as day 4 post-exposure in the highest-dosed marmoset but animals at lower inhaled doses had a protracted disease course where humane study endpoint was not met until as late as day 19 post-exposure. Clinical signs were observed as early as 3 to 4 days post-exposure, including fever, ruffled fur, decreased grooming, and leukocytosis. Clinical signs increased in severity as disease progressed to include decreased body weight, subdued behavior, tremors, and lack of balance. Fever was observed as early as day 2-3 post-exposure in the highest dose groups and hypothermia was observed in several cases as animals became moribund. Infectious virus was found in several key tissues, including brain, liver, kidney, and several lymph nodes. Clinical hematology results included early neutrophilia, lymphopenia, and thrombocytopenia. Key pathological changes included meningoencephalitis and retinitis. Immunohistochemical staining for viral antigen was positive in the brain, retina, and lymph nodes. More intense and widespread IHC labeling occurred with increased aerosol dose.

Conclusion: We have estimated the medial lethal dose of aerosolized EEEV and described the pathology of clinical disease in the marmoset model. The results demonstrate that the marmoset is an animal model suitable for emulation of human EEEV disease in the development of medical countermeasures.

Keywords: Alphavirus; Animal model; Immunity; Nonhuman primate; Pathogenesis.

Fig. 1

Clinical observation scores in marmosets with increasing doses of aerosolized EEEV. Clinical observation parameters included: (1) neurological signs, (2) temperature, (3) appearance, (4) natural behavior, and (5) provoked behavior. Animal behavior was noted and the sum of the score for each parameter was calculated. The values correspond to the highest score obtained for a marmoset per day

Fig. 2

Effect of aerosolized EEEV on total body weight of marmosets. Marmoset weights were recorded every third day during anesthetized physical observation. Weight loss was most evident in marmosets #3 and #4. The disease course, including weight loss, for marmoset #2 followed a more prolonged course, consistent with the lower inhaled dose of EEEV that the animal received. Marmoset #5 failed to show a meaningful decrease in body weight since this animal succumbed to disease on day 4 post-exposure

Fig. 3

Hematological changes in marmosets after aerosolized EEEV challenge. The doses of EEEV to that the marmosets were exposed are indicated in the bottom right of the figure. Grey areas over the graphs correspond to normal value ranges for marmosets. The graphs show the results for (a) white blood cell counts, (b) neutrophils, (c) lymphocytes, (d) monocytes, and (e) platelets in the infected marmosets through time

Fig. 4

Fever response in marmosets after EEEV aerosol challenge. Variation in body temperature is shown for (a) marmoset #1, (b) marmoset #2, (c) marmoset #3, (d) marmoset #4 and (e) marmoset #5. Fever was determined by comparing baseline body temperatures of the marmosets with temperatures measured after aerosol exposure. Baseline temperatures were collected every five minutes from as early as 7 days before challenge. Telemetry collection continued after exposure until study endpoint (up to 28 days post-exposure). Average daily elevations in body temperature and any residual temperature data above 3 SD were used to compute fever duration

Fig. 5

Changes in blood oxygen saturation in the marmosets was measured prior to and following exposure to aerosolized EEEV. Blood oxygen saturation values were determined using a pulse oximeter every 3 days while animals were under anesthesia for physical examination

Fig. 6

Histopathology following exposure to aerosolized EEEV. a Examination of the frontal cortex of the brain revealed the presence of multifocal meningoencephalitis with hemorrhage (HE; magnification, 4×). b Blood vessel in frontal cortex displayed vasculitis with perivascular hemorrhage (arrow) (HE; magnification, 20×). c In the corpus striatum, two neurons (arrows) showed hypereosinophilic perikaryon (cytoplasm) suggesting necrosis. Vasculitis (V) with perivascular hemorrhage was seen in an adjacent vessel and gliosis (asterisk) in the surrounding neuropil (HE; magnification, 40×). d In the pons, three centrally located neurons (arrows) were observed that were shrunken and angular with hypereosinophilic perikaryon and deeply basophilic (hyperchromatic) nuclei (HE; magnification, 60×). Images are from marmoset #4 exposed to 9.76 × 10⁴ PFU of EEEV

Fig. 7

Immunohistochemistry analysis for marmosets exposed to aerosolized EEEV. The image in Panel a from the pons reveals widespread positive antigen staining of neurons in the brain, indicating the presence of EEEV viral RNA(immunohistochemistry; magnification, 40×). Panel b, immunohistochemical staining for the presence of antigen in the retina of marmoset #4. Depicted from the top of the image toward the bottom are: inner limiting membrane, optic fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, and external limiting membrane. The photo receptor layer appears predominantly negative (bottom of image) and the pigment epithelium is barely present in the image. Both images are from marmoset #4 exposed to 9.76 × 104 PFU of EEEV