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. 2013 Jun 15;207(12):1909-21.
doi: 10.1093/infdis/jit061. Epub 2013 Feb 15.

Lethal Crimean-Congo hemorrhagic fever virus infection in interferon α/β receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy

Marko Zivcec  1 , David Safronetz, Dana Scott, Shelly Robertson, Hideki Ebihara, Heinz Feldmann
Affiliations

Lethal Crimean-Congo hemorrhagic fever virus infection in interferon α/β receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy

Marko Zivcec et al. J Infect Dis. .

Abstract

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed viral hemorrhagic fever characterized by rapid onset of flu-like symptoms often followed by hemorrhagic manifestations. CCHF virus (CCHFV), a bunyavirus in the Nairovirus genus, is capable of infecting a wide range of mammalian hosts in nature but so far only causes disease in humans. Recently, immunocompromised mice have been reported as CCHF disease models, but detailed characterization is lacking. Here, we closely followed infection and disease progression in CCHFV-infected interferon α/β receptor knockout (IFNAR(-/-)) mice and age-matched wild-type (WT) mice. WT mice quickly clear CCHFV without developing any disease signs. In contrast, CCHFV infected IFNAR(-/-) mice develop an acute fulminant disease with high viral loads leading to organ pathology (liver and lymphoid tissues), marked proinflammatory host responses, severe thrombocytopenia, coagulopathy, and death. Disease progression closely mimics hallmarks of human CCHF disease, making IFNAR(-/-) mice an excellent choice to assess medical countermeasures.

Keywords: CCHFV; coagulopathy; interferon α/β receptor knockout mice; pathology; proinflammatory response; thrombocytopenia.

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Figures

Figure 1.
Figure 1.
Survival and weight loss among IFNAR−/− mice following infection with Crimean-Congo hemorrhagic fever virus (CCHFV) by different routes. Groups of 6 IFNAR−/− mice were challenged with a high (104 median tissue culture infective doses [TCID50]; A) and a low (102 TCID50; B) dose of CCHFV by the intraperitoneal (i.p.), intramuscular (i.m.), intranasal (i.n.), and subcutaneous (s.c.) routes of inoculation. A, At the high dose of CCHFV, IFNAR−/− mice rapidly succumb to infection and display disease signs, including weight loss (right graph; note, IFNAR−/− mice were weighed daily as a group), ruffled fur, hunched posture, and lethargy. B, At the low dose of CCHFV, IFNAR−/− mice develop similar disease but exhibit a delayed onset and time to death. C, The IFNAR−/− mouse 50% lethal dose for the s.c. route was determined using the standard TCID50 assay to be 0.05 TCID50. Abbreviation: SEM, standard error of the mean.
Figure 2.
Figure 2.
Viremia and organ viral titers of Crimean-Congo hemorrhagic fever virus (CCHFV) in wild-type and IFNAR−/− mice. Groups of 6 mice were inoculated with 200 50% lethal doses (10 median tissue culture infective doses [TCID50]) of CCHFV by the subcutaneous route and sampled at indicated time points after infection. Viral titers were determined by standard TCID50 assay and displayed as standard error about the mean. Dashed line represents the limit of detection of the TCID50 assay (3.16 infectious particles per mg of tissue or µL of blood).
Figure 3.
Figure 3.
Histology (hematoxylin and eosin staining [H&E]) and immunohistochemistry (IHC) analysis of Crimean-Congo hemorrhagic fever virus (CCHFV) in IFNAR−/− mice. Groups of 6 mice were inoculated with 200 50% lethal doses (10 median tissue culture infective doses) by the subcutaneous route and sampled daily after infection. Tissues were stained with hematoxylin and eosin, H&E or a rabbit polyclonal serum directed against the CCHFV nucleoprotein (IHC). Histologic changes were first apparent on day 3 after infection. A, Liver displayed hepatocellular necrosis with infiltration of viable and degenerate neutrophils (solid arrows). The extent of pathologic changes increased over time, resulting in coalescing necrosis with loss of hepatic architecture beginning on day 4 after infection (open arrow).
Figure 3.
Figure 3.
Histology (hematoxylin and eosin staining [H&E]) and immunohistochemistry (IHC) analysis of Crimean-Congo hemorrhagic fever virus (CCHFV) in IFNAR−/− mice. Groups of 6 mice were inoculated with 200 50% lethal doses (10 median tissue culture infective doses) by the subcutaneous route and sampled daily after infection. Tissues were stained with hematoxylin and eosin, H&E or a rabbit polyclonal serum directed against the CCHFV nucleoprotein (IHC). Histologic changes were first apparent on day 3 after infection. A, Liver displayed hepatocellular necrosis with infiltration of viable and degenerate neutrophils (solid arrows). The extent of pathologic changes increased over time, resulting in coalescing necrosis with loss of hepatic architecture beginning on day 4 after infection (open arrow).
Figure 3.
Figure 3.
Histology (hematoxylin and eosin staining [H&E]) and immunohistochemistry (IHC) analysis of Crimean-Congo hemorrhagic fever virus (CCHFV) in IFNAR−/− mice. Groups of 6 mice were inoculated with 200 50% lethal doses (10 median tissue culture infective doses) by the subcutaneous route and sampled daily after infection. Tissues were stained with hematoxylin and eosin, H&E or a rabbit polyclonal serum directed against the CCHFV nucleoprotein (IHC). Histologic changes were first apparent on day 3 after infection. A, Liver displayed hepatocellular necrosis with infiltration of viable and degenerate neutrophils (solid arrows). The extent of pathologic changes increased over time, resulting in coalescing necrosis with loss of hepatic architecture beginning on day 4 after infection (open arrow).
Figure 4.
Figure 4.
Abnormal hematologic, blood chemistry, and coagulation parameters following Crimean-Congo hemorrhagic fever virus (CCHFV) infection in wild-type (WT) and IFNAR−/− mice. A subset of mice were inoculated with 200 50% lethal doses (10 median tissue culture infective doses) by the subcutaneous route and exsanguinated at indicated time points (6 mice were used for hematologic analyses (except 3 IFNAR−/− mice on day 5 after infection), 7 mice were used for coagulation analysis (except 3 IFNAR−/− mice on day 5 after infection), and 1 pool (pools of 6 animal samples on days 1–4 after infection, a pool of 3 animal samples on day 5 after infection) was used for blood chemistry analysis. In contrast to CCHFV-infected WT mice, CCHFV-infected IFNAR−/− mice showed increased plasma alanine aminotransferase (ALT) levels (A), increased aspartate aminotransferase (AST) levels (B), decreased numbers of platelets (PLT; C), increased mean platelet volume (MPV; D), increased serum fibrinogen levels (E), and increased activated partial thromboplastin time (APTT; F). These data suggest that CCHFV infection of IFNAR−/− mice is associated with direct platelet destruction, rather than reduced platelet production. All data were analyzed by 1-way analysis of variance with the Dunnet posttest and are presented as standard error about the mean. ***P < .001 and **P < .01, compared with uninfected controls.
Figure 5.
Figure 5.
Cytokine and chemokine profiles following Crimean-Congo hemorrhagic fever virus (CCHFV) infection in wild-type (WT) and IFNAR−/− mice. Mice (n = 6) were inoculated with 200 50% lethal doses (10 median tissue culture infective doses) of CCHFV by the subcutaneous route and exsanguinated at indicated time points for evaluation of serum cytokine and chemokine levels. CCHFV-infected IFNAR−/− mice displayed marked increases in proinflammatory and chemoattractant molecules (blue graphs), whereas WT mice displayed selective reduction in certain proinflammatory and antiinflammatory molecules (red graphs). All data were analyzed by 1-way analysis of variance with the Dunnet posttest and are presented as standard error about the mean. *P < .05, **P < .01, and ***P < .001, compared with uninfected controls. Abbreviations: CCL2, monocyte chemotactic protein 1; CCL3, macrophage inflammatory protein 1α; CCL5, regulated upon activation, normal T-cell expressed, and secreted; CXCL1, chemokine (C-X-C motif) ligand 1 like; CXCL10, interferon-γ–induced protein 10; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; IFN-γ, interferon γ; IL-1α, interleukin 1α; IL-1β, interleukin 1β; IL-2, interleukin 2; IL-6, interleukin 6; IL-12p70, interleukin 12p70; IL-13, interleukin 13; IL-17, interleukin 17; TNF-α, tumor necrosis factor alpha.
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