Experimental respiratory Marburg virus haemorrhagic fever infection in the common marmoset (Callithrix jacchus)

Abstract

Marburg virus causes a highly infectious and lethal haemorrhagic fever in primates and may be exploited as a potential biothreat pathogen. To combat the infection and threat of Marburg haemorrhagic fever, there is a need to develop and license appropriate medical countermeasures. To determine whether the common marmoset (Callithrix jacchus) would be an appropriate model to assess therapies against Marburg haemorrhagic fever, initial susceptibility, lethality and pathogenesis studies were performed. Low doses of virus, between 4 and 28 TCID50 , were sufficient to cause a lethal, reproducible infection. Animals became febrile between days 5 and 6, maintaining a high fever before succumbing to disease between 8 and 11 days postchallenge. Typical signs of Marburg virus infection were observed including haemorrhaging and a transient rash. In pathogenesis studies, virus was isolated from the animals' lungs from day 3 postchallenge and from the liver, spleen and blood from day 5 postchallenge. Early signs of histopathology were apparent in the kidney and liver from day 3. The most striking features were observed in animals exhibiting severe clinical signs, which included high viral titres in all organs, with the highest levels in the blood, increased levels in liver function enzymes and blood clotting times, decreased levels in platelets, multifocal moderate-to-severe hepatitis and perivascular oedema.

Figure 1

Temperature profile of marmosets after exposure to Marburg virus by the inhalational route. The temperature profile of six animals (represented by six different colours) postchallenge with approximately 10 TCID₅₀ Marburg virus (MARV). Animals were challenged in pairs (red and blue, yellow and green, pink and purple) on three separate occasions to obtain a consistent disease profile.

Figure 2

Virology titres in marmosets postexposure to Marburg virus by the inhalational route. Pairs of marmosets were challenged with approximately 10 TCID₅₀ Marburg virus (MARV) by the inhalational route. At various times postchallenge, two animals were culled. Virology titres in the liver (top left), lung (top right) and spleen (bottom left) were enumerated from organ homogenate. Direct counts for virus numbers in the blood were also measured (bottom right). A total of eight animals over several studies succumbed to infection (terminal time-point). At each time-point, the mean TCID₅₀ count and SEM are plotted.

Figure 3

Selected blood chemistry results and clotting times from marmosets over time after exposure to Marburg virus by the inhalational route. Blood was taken from marmosets prechallenge and at post-mortem at various times postchallenge, and sera were analysed for blood chemistries. (a) Levels of AST, aspartate aminotransferase (filled circle, solid line) and ALT alanine aminotransferase (empty square, dashed line) over time. (b) Levels of ALKP, alkaline phosphatase (filled circle, solid line) and AMYL amylase (empty square, dashed line) over time. (c) Levels of creatinine (filled circle, solid line) and blood urea nitrogen (empty square, dashed line) over time. (d) The clotting time of citrated whole blood was measured; both the activated partial thromboplastin time (aPTT, empty squares, dashed line) and the prothrombin time (PT, filled circles, solid line) were recorded. Data sets include up to 16 values for the prechallenge baseline, n = 2 for time-points at day 1, day 3, day 5 and day 7 postchallenge, and combined data from eight animals that succumbed to infection (terminal).

Figure 4

Marmoset immune response to Marburg infection. Blood was taken from marmosets at post-mortem at various times post-challenge with aerosolized Marburg virus (MARV). Cell numbers (filled shapes and solid lines) are shown against the left Y axis. Cytokine concentrations (empty shapes, dashed lines) are shown against the right Y axis. Mean of duplicate marmosets plus SEM shown.

Figure 5

Haematoxylin and eosin (H&E)-stained histology images from marmosets after succumbing to infection with Marburg virus by the inhalational route. (a) Liver. Multiple hepatocellular necrotic foci with haemorrhages (arrows). Insert: necrosis and inflammatory cell infiltrates in the portal area. (b) Spleen. Lymphoid follicle showing significant lymphoid depletion, particularly in the centre of the follicle. (c) Kidney. Glomerular capillaries showing endothelial necrosis and thrombosis. The Bowman's capsule is filled with a protein rich exudate and cellular debris. (d) Kidney. Congestion, intertubular haemorrhages and mild inflammatory infiltrates. Degeneration of tubular epithelial cells and protein casts in the tubular lumina. Insert: haemosiderin in the cytoplasm of tubular epithelial cells. (e) Lung. Marked perivascular oedema (arrows). (f) Lung. Diffuse alveolar haemorrhage and necrosis of alveolar septal walls.

Figure 6

Electron microscopy images from marmoset lung after succumbing to infection with Marburg virus by the inhalational route. (a) SEM of a group of alveoli showing filling with extravasated red blood cells and fibrin deposition. (b) transmission electron microscopy (TEM) of an infected macrophage within an alveolar space. The macrophage contains numerous vacuoles, some of which can be seen to harbour developing virion particles. (c) TEM of the alveolar space adjacent to the infected macrophage showing mature viral particles (arrows) (d, e) Higher magnification TEMs of the developing virion particles within macrophage vacuoles. Scale bars: a – 20 μm; b – 2 μm; c and d – 500 nm; e – 100 nm

Figure 7

A range of staining techniques applied to different marmoset organs after infection with Marburg virus by the inhalational route. a–d are from immunohistochemistry (IHC) staining with an anti-Marburg antibody that appears brown. (a) IHC of liver section 7 days after challenge. (b) IHC of spleen section 3 days after challenge. (c) IHC of kidney section at the terminal point of challenge. (d) IHC of thymus section at the terminal point postchallenge. (e) TUNEL (dark brown) staining for apoptosis of a liver section at day 7 postchallenge. (f) TUNEL (dark brown) staining for apoptosis of a spleen section at day 3 postchallenge. (g) Martius Scarlet Blue staining for fibrin of kidney section at the terminal point postchallenge. New fibrin stains red and old fibrin stains blue. (h) Macchiavello staining for inclusion bodies (stained red) of liver section at the terminal point postchallenge.