Crimean-Congo Hemorrhagic Fever Mouse Model Recapitulating Human Convalescence

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is a cause of severe hemorrhagic fever. Its tick reservoir and vector are widely distributed throughout Africa, Southern and Eastern Europe, the Middle East, and Asia. Serological evidence suggests that CCHFV can productively infect a wide variety of species, but only humans develop severe, sometimes fatal disease. The role of the host adaptive immunity in control or contribution to the severe pathology seen in CCHF cases is largely unknown. Studies of adaptive immune responses to CCHFV have been limited due to lack of suitable small animal models. Wild-type mice are resistant to CCHFV infection, and type I interferon-deficient mice typically develop a rapid-onset fatal disease prior to development of adaptive immune responses. We report here a mouse model in which type I interferon-deficient mice infected with a clinical isolate of CCHFV develop a severe inflammatory disease but ultimately recover. Recovery was coincident with development of CCHFV-specific B- and T-cell responses that were sustained for weeks postinfection. We also found that recovery from a primary CCHFV infection could protect against disease following homologous or heterologous reinfection. Together this model enables study of multiple aspects of CCHFV pathogenesis, including convalescence, an important aspect of CCHF disease that existing mouse models have been unsuitable for studying.IMPORTANCE The role of antibody or virus-specific T-cell responses in control of acute Crimean-Congo hemorrhagic fever virus infection is largely unclear. This is a critical gap in our understanding of CCHF, and investigation of convalescence following severe acute CCHF has been limited by the lack of suitable small animal models. We report here a mouse model of CCHF in which infected mice develop severe disease but ultimately recover. Although mice developed an inflammatory immune response along with severe liver and spleen pathology, these mice also developed CCHFV-specific B- and T-cell responses and were protected from reinfection. This model provides a valuable tool to investigate how host immune responses control acute CCHFV infection and how these responses may contribute to the severe disease seen in CCHFV-infected humans in order to develop therapeutic interventions that promote protective immune responses.

Keywords: Crimean-Congo hemorrhagic fever virus; adaptive immunity; animal models; inflammation; pathogenesis.

FIG 1

Adult IFNAR^−/− mice inoculated subcutaneously with CCHFV strain Hoti develop severe disease and high viral RNA loads. Eight- to 12-week-old IFNAR^−/− mice were inoculated with indicated dose of either CCHFV strain IbAr 10200 or strain Hoti via a single subcutaneous injection to the subscapular region. Mice were weighed daily (A) and monitored for clinical disease. Mice were humanely euthanized according to criteria described in Materials and Methods (B). n = 6 per group. (C and D) Eight- to 12-week-old IFNAR^−/− mice were inoculated with 1 TCID₅₀ of CCHFV strain Hoti. At the indicated time points, viral RNA loads in the serum (C) and tissues (D) were quantified by qRT-PCR. n = 5 to 8 per group. The dashed line indicates the limit of detection.

FIG 2

IFNAR^−/− mice inoculated with CCHFV Hoti exhibit liver pathology. IFNAR^−/− mice were inoculated with 1 TCID₅₀ of CCHFV strain Hoti. At the indicated time points, levels of aspartate aminotransferase (AST) (A) and alanine aminotransferase (ALT) (B) in the blood were quantified using Preventive Care disks in a Vetscan2 analyzer. Statistical comparison to mock-infected mice was performed using an ordinary one-way analysis of variance (ANOVA) with the Holm-Sidak multiple-comparison test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. n = 5 to 9 per group. Formalin-fixed sections of liver tissue were stained with hematoxylin and eosin (C) and evaluated for pathology. To identify viral antigen, formalin-fixed sections were stained with rabbit anti-CCHFV NP (D). Representative images are shown at ×10 magnification (C and D) and ×40 magnification (insets in panel D). Black arrows indicate lesions at 14 and 28 dpi.

FIG 3

IFNAR^−/− mice inoculated with CCHFV Hoti exhibit severe spleen pathology. IFNAR^−/− mice were inoculated with 1 TCID₅₀ of CCHFV strain Hoti. At the indicated time points, the spleen was collected and fixed with formalin. Formalin-fixed sections of spleen tissue were stained with hematoxylin and eosin (A) and evaluated for pathology. To identify viral antigen, formalin-fixed sections were stained with rabbit anti-CCHFV NP (B). Representative images are shown at ×10 magnification (A and B) and ×40 magnification (inset B). Black arrows at 4 dpi indicate white pulp necrosis, and those at 28 dpi indicate a hyperplastic follicle.

FIG 4

IFNAR^−/− mice inoculated with CCHFV Hoti develop an inflammatory immune response to the infection. IFNAR^−/− mice were inoculated with 1 TCID₅₀ of CCHFV strain Hoti. At the indicated time points, serum was collected and irradiated, and cytokine levels were quantified with a 23-plex mouse cytokine kit. Statistical comparison to mock-infected mice was performed using an ordinary one-way ANOVA with the Holm-Sidak multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n = 5 to 8 per group. Horizontal bars indicate the mean.

FIG 5

CCHFV Hoti infection results in expansion of neutrophils and inflammatory macrophages. IFNAR^−/− mice were inoculated with 1 TCID₅₀ of CCHFV strain Hoti. At the indicated time points, the spleen was collected and stained with fluorescently conjugated antibodies for flow cytometry. Cells were gated to exclude debris, doublets, and nonviable cells. Neutrophils (A), inflammatory macrophages (B), Ly6C^low macrophages (C), peripheral dendritic cells (pDCs) (D), and classical dendritic cells (cDCs) (E) were identified according to the gating strategy outlined in the text (data not shown). Horizontal bars indicate the mean. Samples from mock-infected mice were collected at each time point and pooled for analysis. n = 24 for mock, 6 each for days 3, 8, and 14 p.i., and 12 for day 28 p.i. Statistical comparison to mock-infected mice was performed using an ordinary one-way ANOVA with the Holm-Sidak multiple-comparison test for cell numbers and percentages and two-way ANOVA with Dunnett’s multiple-comparison test for median fluorescent intensity of activation markers. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 6

IFNAR^−/− mice inoculated with CCHFV Hoti develop CCHFV-specific B- and T-cell responses. IFNAR^−/− mice were inoculated with 1 TCID₅₀ of CCHFV strain Hoti. At the indicated time points, serum was collected, and the amount of CCHFV-specific antibody was quantified by ELISA (A). The 405-nm absorbance of a 1:1,600 dilution of serum is shown. The dashed line indicates background absorbance determined from wells receiving no serum. n = 5 to 8 per group. Neutralizing capacity of serum was quantified by focus reduction neutralization test (B). FRNT₅₀ was calculated by nonlinear regression, and the 95% confidence interval is shown. n = 5 mice per time point. Each serum sample was measured in triplicate, and error bars indicate standard deviation. CCHFV-specific T-cell responses in the spleen were quantified using an IFN-γ ELIspot (C). Cells were stimulated with overlapping peptides derived from the CCHFV nucleoprotein pooled at 18 to 25 peptides per pool. Horizontal bars indicate the mean. Statistical comparison to unstimulated splenocytes was performed using a two-way ANOVA with Dunnett’s multiple-comparison test at each respective time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 7

Recovery from primary CCHFV Hoti infection protects against heterologous and homologous reinfection. Eight- to 12-week-old IFNAR^−/− mice were mock infected or inoculated with 1 TCID₅₀ of CCHFV strain Hoti, and mice were allowed to recover from the infection. At 9.5 weeks later, mice were either mock infected or infected with 10 TCID₅₀ of CCHFV strain Hoti or strain UG3010 via i.p. injections. Mice were weighed daily, monitored for disease, and humanely euthanized according to criteria listed in Materials and Methods (A and B). n = 8 to 12 per group. On day +4, a subset of mice were euthanized, and the liver, spleen, and kidney were collected for evaluation of viral RNA loads by qRT-PCR (C). Horizontal bars indicate the mean, and dashed lines indicate the limit of detection. Statistical comparison to mock-infected mice was performed using an ordinary one-way ANOVA with the Holm-Sidak multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.