Animal Models of Emerging Tick-Borne Phleboviruses: Determining Target Cells in a Lethal Model of SFTSV Infection

Abstract

The pathogenesis of clinical manifestations caused by newly emerging tick-borne phleboviruses [i.e., Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV)], such as severe thrombocytopenia and lymphocytopenia, are not yet fully understood. In the present study, to establish an animal model mimicking the profile of fatal human cases, we examined the susceptibilities of adult mice from 12 strains, aged mice from two strains, and cynomolgus macaques to SFTSV and/or HRTV infections. However, none of these immunocompetent animals developed lethal diseases after infection with SFTSV or HRTV. Thus, we tested a lethal animal model of SFTSV infection using interferon-α/β receptor knock-out (IFNAR^-/-) mice to identify the target cell(s) of virus infection, as well as lesions that are potentially associated with hematological changes. IbaI-positive macrophages and Pax5-positive immature B cells overlapped with SFTSV-positive cells in the spleen and lymph nodes of IFNAR^-/- mice, and IbaI-SFTSV-double positive cells were also observed in the liver and kidney, thereby suggesting crucial roles for macrophages in the pathogenesis of SFTSV infection in mice. In the mandibular lymph nodes and spleens of infected mice, we observed extensive necrosis comprising B220-positive B cells, which may be associated with severe lymphocytopenia. The results of this study suggest a resemblance between the IFNAR^-/- mouse model and lethal infections in humans, as well as roles for multiple cells during pathogenesis in mice.

Keywords: aged mouse; disease modeling; heartland virus; immunocompromised mouse; mouse; nonhuman primate; severe fever with thrombocytopenia syndrome virus.

FIGURE 1

Clinical manifestations in IFNAR^-/- mice infected with SFTSV. Groups of four IFNAR^-/- mice were infected with a high dose (10⁵ TCID₅₀/mouse) or low dose (10² TCID₅₀/mouse) of the SFTSV strain SD4, intradermally (i.d.), intraperitoneally (i.p.), intramuscularly (i.m.), or subcutaneously (s.c.). The mice were monitored daily to assess survival (A) and body weight (B) and results until 6 days post inoculation (dpi) were shown. The blood from the infected mice euthanized because they reached the humane endpoint and the uninfected mice euthanized at 6 dpi were subjected to virus titration (C) and hematological examinations, i.e., lymphocyte count (D), platelet count (E), and mean platelet volume (F). All values are indicated as means ± SD. ^∗P < 0.05, compared with uninfected controls.

FIGURE 2

Histopathological observations of tissues of the IFNAR^-/- mice infected with SFTSV. The spleen, lymph nodes, bone marrow, and livers of SFTSV-infected and uninfected mice were used for histopathologic observations. Losses of the structure with massive apoptosis were observed in hematoxylin and eosin-stained tissues (A); Magnified images of pyknosis and karyorrhexis observed in the necrotic lesions are indicated at the top right corner of the infected spleen and lymph node. The increased myeloid/erythroid ratio in the bone marrow of infected mice was observed (B); A magnified image of moderate necrosis collocating with edema and fibrin is shown at the top right corner. Colocalization of B220-positive cells with necrotic lesions in the spleen and lymph node (C) as well as IbaI-positive cells with enlarged cytoplasm in the liver (D) were visualized by immunohistochemistry.

FIGURE 3

SFTSV infection in tissues from IFNAR^-/- mice infected with SFTSV. The spleen (A), lymph node (B), liver (C), and kidney (D) of SFTSV-infected mice were stained with antibodies raised against SFTSV N antigen. Antigen-positive monocyte-like cells (magnified images at the top), lymphocyte-like cells (middle), and reticular cells (bottom) were morphologically identified in the sample tissues.

FIGURE 4

Colocalization of host cell markers and SFTSV antigen in SFTSV-infected tissues from IFNAR^-/- mice. The spleen (A) lymph node (B), liver (C), and kidney (D) of the SFTSV-infected mice were subjected to double staining with anti-SFTSV N antibodies and host cellular markers: CD3e (T cells), IbaI (macrophages), Pax5 (immature B cells), and gp36 (reticular cells). Cells stained with both SFTSV and host cellular markers are indicated by arrowheads, and the phagocytosis of an infected cell by an IbaI-positive macrophage is indicated by an arrow.