A cynomolgus macaque model for Crimean-Congo haemorrhagic fever

Abstract

Crimean-Congo haemorrhagic fever (CCHF) is the most medically significant tick-borne disease, being widespread in the Middle East, Asia, Africa and parts of Europe ¹ . Increasing case numbers, westerly movement and broadly ranging case fatality rates substantiate the concern of CCHF as a public health threat. Ixodid ticks of the genus Hyalomma are the vector for CCHF virus (CCHFV), an arbovirus in the genus Orthonairovirus of the family Nairoviridae. CCHFV naturally infects numerous wild and domestic animals via tick bite without causing obvious disease^2,3. Severe disease occurs only in humans and transmission usually happens through tick bite or contact with infected animals or humans. The only CCHF disease model is a subset of immunocompromised mice^4-6. Here, we show that following CCHFV infection, cynomolgus macaques exhibited hallmark signs of human CCHF with remarkably similar viral dissemination, organ pathology and disease progression. Histopathology showed infection of hepatocytes, endothelial cells and monocytes and fatal outcome seemed associated with endothelial dysfunction manifesting in a clinical shock syndrome with coagulopathy. This non-human primate model will be an invaluable asset for CCHFV countermeasures development.

Fig. 1 |. Clinical parameters for CCHFV-infected cynomolgus macaques.

Parameters were measured for each animal on days of examination and at time of euthanasia (n = 4 animals per inoculation route). Graphs represent a single biological experiment. a, Clinical score, with euthanasia mandated at a score of 35. b, Survival curves. c, Viral RNA load in whole blood of animals, by infection route, standardized to TCID₅₀ equivalents of the stock challenge virus RNA per ml of whole blood. d, Platelet counts. e, Total protein levels. f, Albumin levels. g, Aspartate aminotransferase (AST) levels. h, Alanine aminotransferase (ALT) levels. i, Activated partial thromboplastin time (APTT). j, Thrombin time; a 60-second clotting time is the preset limit to this test, as surpassed by 4 of 12 animals. Data are shown as individual points with lines representing the mean response by inoculation route, except in the case of panel b, which depicts survival based on inoculation route. Statistical analysis by one-way analysis of variance and a Tukey’s multiple comparison test defines significance between inoculation routes on given examination days. All statistically significant differences represent a difference between either the intravenous- or SC/IV-inoculated group (as designated by colour) and the subcutaneous-inoculated group. Analysis was performed only through to 7 dpi due to reduced group numbers after this point. *P<0.05, **P< 0.005, ***P<0.001. Individual P values are as follows (95% confidence interval): a, SC/IV 4 dpi = 0.0019, intravenous 5 dpi = 0.0174, SC/IV 5 dpi = 0.0013, intravenous 6 dpi = 0.0273, SC/IV 6 dpi = 0.0011, SC/IV 7 dpi = 0.0310; c, intravenous 3 dpi = 0.0265, SC/IV 3 dpi = 0.0032; d, intravenous 5 dpi = 0.0491, SC/IV 4 dpi = 0.0459, intravenous 7 dpi = 0.0040, SC/IV 7 dpi = 0.0025; e, SC/IV 5 dpi = 0.0404, SC/IV 7 dpi = 0.0155; g,SC/IV3dpi = 0.0072, SC/IV 5 dpi = 0.0130; h, SC/IV 3 dpi = 0.0487, SC/IV 5 dpi = 0.0047; i,intravenous3dpi = 0.0073, SC/IV 3 dpi = 0.001, SC/IV 5 dpi = 0.0050, SC/IV 7 dpi = 0.0368; j, SC/IV 3 dpi = 0.0401, SC/IV5dpi = 0.0001,intravenous7dpi = 0.0222, SC/IV 7 dpi = 0.0013. IV, intravenous; SC, subcutaneous.

Fig. 2 |. Profile of immune factors by infection route in CCHFV-infected cynomolgus macaques.

Concentrations of cytokine or chemokine levels were determined in serum samples collected during clinical exams using a multiplex NHP cytokine detection kit. a, IL-6. b, IL-10. c, IL-1β. d, IL-1RA. e, MCP-1. f, MIP-1β. g, IL-15. h, IL-17A. Examination days depicted represent the onset of symptoms (3 dpi), terminal disease (7dpi) or onset of convalescence (9 dpi). Results are of a single biological experiment with n = 4 animals per inoculation route. Data points are graphed individually with standard error of the mean for each inoculation route. Statistical analysis by one-way analysis of variance and a Tukey’s multiple comparison test showed no significance between inoculation routes on given examination days.

Fig. 3 |. Histopathology, immunohistochemistry and in situ hybridization (viral genomic RNA) of the liver from CCHFV-infected cynomolgus macaques.

a-c, Haematoxylin and eosin (H&E) ×100, inset ×400. The panels show multifocal, minimal lymphohistiocytic infiltrates (a); multifocal, minimal lymphohistiocytic infiltrates (b); and multifocal mild-moderate lymphohistiocytic infiltrates (c). Scale bars,50 μm (main),20 \μm (inset). d-f, Immunohistochemistry (IHC) ×400. The panels show no immunoreactivity (d); rare immunoreactivity in hepatocytes, Kupffer cells and endothelial cells (e); and scattered to moderate immunoreactivity in hepatocytes, Kupffer cells and endothelial cells (f). Scale bars, 20 μm. g-i, In situ hybridization (ISH) depicting viral RNA x100. The panels show no immunoreactivity (g); moderate immunoreactivity in hepatocytes, Kupffer cells and endothelial cells (h); and moderate-numerous immunoreactivity in hepatocytes, Kupffer cells and endothelial cells (i). Scale bars,50 μm. Figures are representative of all animals within the inoculation group (n = 4 animals).

Fig. 4 |. Profile of CCHF disease in cynomolgus macaques.

As with human disease, the macaque disease progression may be divided into four periods, with viraemia escalating as platelets drop in the presymptomatic and prehaemorrhagic period, followed by a rise in liver enzymes and elongation in clotting times in the haemorrhagic period. These clinical signs gradually return to normal as the animals enter convalescence.