Computed Tomography Imaging for Monitoring of Marburg Virus Disease: a Nonhuman Primate Proof-Of-Concept Study

Abstract

Marburg virus (MARV) is a highly virulent zoonotic filovirid that causes Marburg virus disease (MVD) in humans. The pathogenesis of MVD remains poorly understood, partially due to the low number of cases that can be studied, the absence of state-of-the-art medical equipment in areas where cases are reported, and limitations on the number of animals that can be safely used in experimental studies under maximum containment animal biosafety level 4 conditions. Medical imaging modalities, such as whole-body computed tomography (CT), may help to describe disease progression in vivo, potentially replacing ethically contentious and logistically challenging serial euthanasia studies. Towards this vision, we performed a pilot study, during which we acquired whole-body CT images of 6 rhesus monkeys before and 7 to 9 days after intramuscular MARV exposure. We identified imaging abnormalities in the liver, spleen, and axillary lymph nodes that corresponded to clinical, virological, and gross pathological hallmarks of MVD in this animal model. Quantitative image analysis indicated hepatomegaly with a significant reduction in organ density (indicating fatty infiltration of the liver), splenomegaly, and edema that corresponded with gross pathological and histopathological findings. Our results indicated that CT imaging could be used to verify and quantify typical MVD pathogenesis versus altered, diminished, or absent disease severity or progression in the presence of candidate medical countermeasures, thus possibly reducing the number of animals needed and eliminating serial euthanasia. IMPORTANCE Marburg virus (MARV) is a highly virulent zoonotic filovirid that causes Marburg virus disease (MVD) in humans. Much is unknown about disease progression and, thus, prevention and treatment options are limited. Medical imaging modalities, such as whole-body computed tomography (CT), have the potential to improve understanding of MVD pathogenesis. Our study used CT to identify abnormalities in the liver, spleen, and axillary lymph nodes that corresponded to known clinical signs of MVD in this animal model. Our results indicated that CT imaging and analyses could be used to elucidate pathogenesis and possibly assess the efficacy of candidate treatments.

Keywords: Marburg virus; computed tomography; filovirus; medical imaging; viral pathogenesis.

FIG 1

Study design. A total of 6 rhesus monkeys were imaged 21, 14, and 7 days prior to and on the day of euthanasia after MARV exposure. MARV, Marburg virus; CT, computed tomography; BL, baseline; DE, day of euthanasia.

FIG 2

Qualitative CT imaging without contrast (rhesus monkey). (Top) Coronal view of bilateral axillary adenopathy after exposure to MARV via the intramuscular route. (A) Baseline scan shows normal axillary nodes bilaterally. (B) Scan on day of euthanasia, 9 days after exposure, shows right axillary adenopathy and diffuse infiltration of axillary fat (white arrowhead) and left axillary lymphadenopathy (white arrow). (Bottom) Axial views of changes in pancreas following MARV exposure. (C) Baseline scan shows normal pancreas (encircled with green line) and retroperitoneum. (D) Scan on day of euthanasia, 8 days after exposure, shows replacement of the area of the pancreas with low-radiodensity tissue (encircled with green line), which proved to be pancreatic exocrine atrophy at necropsy. BL, baseline; DE, day of euthanasia.

FIG 3

Quantitative CT imaging results (rhesus monkey liver). Comparison of baseline scans and those acquired on the day of euthanasia shows hepatomegaly and decreased radiodensity after MARV exposure. (A) Coronal CT images and 3D volume renderings of the liver, based on segmentation masks. Regions of interest segmenting the liver (green borders) were automatically generated using a machine-learning-based algorithm. Data from 3 baseline imaging sessions were highly reproducible. (B) Changes in liver volume. (C) Changes in liver radiodensity (expressed in Hounsfield units [HU]). (D) Gross pathology of the liver on the day of euthanasia. The liver was markedly enlarged, with rounded edges, pale tan-yellow color, and a greasy and friable consistency (lipidosis and necrosis). ns, not significant; DE, day of euthanasia (8 days postexposure).

FIG 4

Quantitative CT imaging results (rhesus monkey spleen). Comparison of baseline scans and those acquired on the day of euthanasia shows splenomegaly and decreased radiodensity after MARV exposure. (A) Coronal CT images and 3D volume renderings of the spleen, based on segmentation masks. Regions of interest segmenting the spleen (green borders) were automatically generated using a machine-learning-based algorithm. Data from 3 baseline imaging sessions were highly reproducible. (B) Changes in spleen volume. (C) Changes in spleen radiodensity (expressed as Hounsfield units [HU]). (D) Gross pathology of the spleen on the day of euthanasia. The spleen was moderately enlarged, with rounded edges and a friable consistency. ns, not significant; DE, day of euthanasia (8 days postexposure).

FIG 5

Liver histopathology images (rhesus monkey) on day of euthanasia (DE), 8 days after MARV exposure. (A) Tissue stained with hematoxylin and eosin (H&E) shows multifocal to coalescing hepatocellular degeneration and necrosis that affected approximately 20% of hepatocytes and diffuse mild to moderate microvesicular lipid-type cytoplasmic vacuolation of hepatocytes (black arrows). (B) IHC detection of MARV glycoprotein (brown) in hepatocytes, Kupffer cells, and sinusoids. (C) TEM showing abundant mature MARV particles (yellow box) between hepatocytes. (D) ISH results, demonstrates abundant MARV genome (darker pink) in viable and degenerate hepatocytes and Kupffer cells.