Temporal Characterization of Marburg Virus Angola Infection following Aerosol Challenge in Rhesus Macaques

Abstract

Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, 15 rhesus macaques were sequentially sacrificed following aerosol exposure to the MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 postexposure (p.e.). The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 p.e. High (>10(7) PFU/ml) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 p.e. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as interleukin 10 (IL-10) and IL-5 and late-stage increases in Th1 cytokines such as IL-2, IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model.

Importance: In this study, we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following aerosol exposure.

FIG 1

Body temperature and viremia. (A) Febrile illness as evidenced by changes in rectal body temperature. (B) Viremia was determined by plaque assay and qRT-PCR. qRT-PCR values are represented as PFU/ml equivalents calculated from viral genome copies. Each data point represents the average value for all samples that were analyzed at that time point.

FIG 2

Tissue-associated MARV titers. Averaged tissue viral titers from qRT-PCR (PFU/ml equivalents calculated from viral genomes copies) (A) and averaged tissue viral titers from plaque assay per tissue type (B) demonstrate virus dissemination throughout MARV disease course. (C and D) Composites of all tissue-associated viral titers for each animal grouped by sacrifice day, as determined by qRT-PCR (C) and plaque assay (D). Ax, axillary; Ing, inguinal; LLoQ, lower limit of quantification; LN, lymph node; Mand, mandibular; Mes, mesenteric; TB, tracheobronchial; ULoQ, upper limit of quantification.

FIG 3

Hematology and coagulation profiles. Platelet deregulation (A), increases in clotting times (B), and the presence of D-dimers (C) illustrate a disruption in normal coagulation function. (D) Red blood cell and hematocrit remained relatively normal throughout the study, with increases attributed to splenic contraction and whole-body dehydration. Increasing total white blood cell counts (E) and neutrophil and lymphocyte counts (F) demonstrate progressive leukocytosis and lymphocytosis. Dashed lines represent ranges for RBC and lymphocytes on respective graphs. Dotted lines represent range values for hematocrit and neutrophils on respective graphs. Each data point represents the average value for all samples that were analyzed at that time point.

FIG 4

Clinical chemistry. Increased concentrations of creatinine (A) and blood urea nitrogen (B) confirm azotemia. A drop in total protein and albumin concentrations (C), along with increases in aspartate transaminase (AST) and alanine transaminase (ALT) (D), are associated with liver damage. Dashed and dotted lines represent normal range values. Each data point represents the average value for all samples that were analyzed at that time point.

FIG 5

Cytokines and chemokines. Data are the change (fold) over baseline (dotted lines) for selected recruitment and proinflammatory cytokines and chemokines. Changes indicate a Th1 immune response late in disease. Each data point represents the average value for all samples that were analyzed at that time point.

FIG 6

Pathology. (A) Gross pathology of tracheobronchial lymph nodes on day 3 p.e. (arrows) shows a mild increase in size and normal coloration. (B) Gross pathology image of tracheobronchial lymph nodes on day 9 p.e. (arrows) shows red to black (hemorrhage) discoloration and enlargement (approximately five times normal size). (C) Hematoxylin and eosin (HE) stain (magnification of ×100) of normal tracheobronchial lymph nodes on day 1 p.e. (D) HE stain (magnification of ×100) of tracheobronchial lymph node on day 3 p.e. shows sinuses (asterisks) that are severely expanded by a high number of macrophages, confirmed with anti-macrophage antibody 387 (MAC387) (inset), that surround and widely separate follicles (arrow). (E) HE stain (magnification of ×100) of tracheobronchial lymph nodes on day 9 p.e. demonstrates diffuse necrosis, edema, and hemorrhage. Gross liver images from days 1 (F) and 7 (G) p.e. show the dramatic change to yellow discoloration late in disease.

FIG 7

Pathology. No gross changes in the lungs are evident from day 1 p.e. (A) and day 9 p.e. (B). (C) HE stain of the lungs on day 9 p.e. (magnification of ×200) shows mild increases in alveolar histiocytes (arrow) and fibrin thromboemboli within blood vessels (asterisk) compared with normal day 1 p.e. lung (inset; 100× HE). (D) Axillary haired skin with mild macular rash day 9 p.e. (E) HE stain (magnification of ×100) of day 9 p.e. axillary haired skin that contains superficial blood vessels with perivascular inflammation (arrows) and no hemorrhage in the dermis. (F) Higher magnification (400× HE) of superficial dermal blood vessel shows perivascular inflammation and fibrin within blood vessels (arrow). Caspase 3 IHC (magnification of ×100) staining of the tracheobronchial lymph node on days 3 (G), 5 (H), 7 (I), and 9 (J) p.e. illustrate the progressive and profound apoptosis of lymphocytes in the germinal centers of lymphoid follicles. Caspase 3 IHC staining (magnification of ×100) of the spleen on days 3 (K), 5 (L), and 7 (M) p.e. illustrates occasional to focal apoptosis in germinal centers of splenic corpuscles that progress to diffuse apoptosis on day 9 p.e. (N).