Severe fever with thrombocytopenia syndrome virus strain SD4 was provided by the Chinese Center for Disease Control and Prevention and HRTV strain Mo4 was kindly provided by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) from the arthropod-borne virus reference collection at the University of Texas Medical Branch (UTMB). These strains were propagated in a human hepatocyte cell line, i.e., human hepatocellular carcinoma cells (Huh7), for 5 days and the supernatant was centrifuged twice to remove any debris. The Huh7 cell line was kindly provided by Dr Yoshiharu Matsuura, Osaka University, and it was maintained in Dulbecco’s Modified Eagle Medium supplemented with 2% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin (Life Technologies), and 10 μg/ml MycoKill AB (GE Healthcare). The virus infectivity titers in blood were determined as the median tissue culture infectious dose (TCID₅₀) detected by using an indirect immunofluorescent antibody assay with mouse immune ascitic fluids generated against SFTSV or HRTV, which were provided by Dr. Robert Tesh, UTMB (Matsuno et al., 2013).

Groups of four 6- to 12-week-old mice from inbred strains (i.e., 129S1/svlmJ, A/J, C57BL/6J, CAST/EiJ, DBA/1J, DBA/2J, FVB/NJ, NZBWF1/J, and SIL/J), recombinant inbred strains (i.e., BXD68/RwwJ and BXD34/TyJ), an outbred strain (i.e., ICR CD-1) (obtained from Jackson Laboratory), IFNAR^-/- C57BL/6 (breeding pairs kindly provided by Dr. Genhong Cheng, University of California Los Angeles), and aged (10 to >20 months) 129S1/svlmJ and C57BL/6J strains (obtained from Jackson Laboratory and kept in-house) were inoculated with a high dose (10⁵ TCID₅₀/animal) or a low dose (10² TCID₅₀/animal) of SFTSV SD4 intradermally (i.d.), intraperitoneally (i.p.), intramuscularly (i.m.), or subcutaneously (s.c.). The animals were monitored for 14 days after challenge. Animals that reached the humane endpoint were euthanized and terminally bled by cardiac puncture. To determine the median mouse lethal dose (MLD₅₀), groups of four IFNAR^-/- C57BL/6 mice were infected with serial 10-fold dilutions of SFTSV SD4 and observed for 2 weeks.

Two groups of two cynomolgus macaques (2 to 3 kg) were inoculated s.c. with 10⁶ TCID₅₀ of SFTSV SD4 or HRTV Mo4. At 1, 3, 5, 8, 11, and 14 days post inoculation (dpi), blood was drawn and clinical exams were performed on the anesthetized animals.

Hematological parameters were analyzed using EDTA-treated whole blood with a HemaVet 950FS1 laser-based hematology analyzer (Drew Scientific). The parameters of infected IFNAR^-/- mice and uninfected mice euthanized were compared statistically by Mann-Whitney test on GraphPad Prism v6.0h (GraphPad Software).

Tissues fixed with neutral-buffered formalin (10% v/v) were processed and embedded in paraffin according to standard procedures. The embedded tissues were sectioned at 5 μm and dried overnight at 42°C before staining with hematoxylin and eosin. To detect the viral antigen by immunohistochemistry, a rabbit polyclonal antiserum against SFTSV N protein (kindly provided by Dr. Shigeru Morikawa, National Institute of Infectious Diseases, Japan) was used as the primary antibody. Different types of cells were identified immunohistochemically as follows: white blood cells [CD45R (B220)], immature B cells (Pax5), T cells (CD3e), macrophages (IbaI), and reticular cells (gp36), which were stained with Rat anti-CD45R (BD Biosciences), Rabbit mAb PAX5 (Cell Signaling), Goat anti-CD3-𝜀 (Santa Cruz), Rabbit anti-Iba-1 (Wako), and Hamster anti-Podoplanin (Novusbio), respectively. Each antigen was visualized with Envision++ system HRP Rabbit (DAKO), Simple stain AP Rabbit, Simple Stain MAX PO Rat (Nichirei), ImmPress HRP anti-goat IgG (Vector), or Biotinylated anti-Hamster IgG (Vector) with SAB-PO (Nichirei) in an appropriate combination according to the manufacturer’s protocol.

All infectious work with SFTSV or HRTV was performed in a high containment facility at the Rocky Mountain Laboratories (RML), Division of Intramural Research (DIR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). The work was approved by the RML Institutional Biosafety Committee (IBC) at biosafety level 3 (BSL3).

All of the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the RML, and performed following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) by certified staff in an AAALAC-approved facility, following the guidelines and basic principles of the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals.

In order to compare the susceptibility of various laboratory mouse strains and different age groups of mice to SFTSV infection, we inoculated groups of four mice from nine inbred mouse strains, two recombinant inbred strains, and one outbred strain with a high dose (10⁵ TCID₅₀/animal) or a low dose (10² TCID₅₀/animal) of SFTSV SD4, i.d., i.p., i.m., or s.c. (Table 1). In addition, increased age is a significant risk factor for the severity of disease in SFTS, especially among those aged >60 years (Xiong et al., 2012; Ding et al., 2014; Takahashi et al., 2014), therefore, aged (12–24 months) mouse models based on two different inbred strains (129S1/svlmJ and C57BL6/J), which represented humans aged >60 years (Flurkey et al., 2006), were also infected with SFTSV. None of the mice challenged in this study developed lethal diseases and no significant lesions were found in histological examinations of the mice (Table 1). Twelve-month-old C57BL/6 mice and >20-month-old C57BL/6 mice infected with SFTSV via s.c. route, led to only moderate weight loss and all of them survived until 14 dpi.

It has been reported that SFTSV infection causes mild illness with thrombocytopenia and leukocytopenia in rhesus macaques. Therefore, in order to investigate whether pathogenic tick-borne phleboviruses induce visible or severe disease in cynomolgus macaques, two groups of two macaques were infected subcutaneously with 10⁶ TCID₅₀ of SFTSV SD4 or HRTV Mo4. All four macaques infected with SFTSV or HRTV exhibited no visible clinical signs, except one animal infected with SFTSV had a temporarily decreased platelet count. Moreover, in all four of the infected animals, no viruses were detected from blood during the examination period (at 1, 3, 5, 8, 11, and 14 dpi) and no macroscopic lesions were found at 14 dpi.

None of the immunocompetent animals tested in this study developed severe/fatal disease after SFTSV infection, so we evaluated the sensitivity and lethality of IFNAR^-/- mice via several infection routes and characterized the serum viral load and hematological status of the mice after SFTSV infection. The groups of IFNAR^-/- mice inoculated with different doses of SFTSV SD4 via different inoculation routes exhibited severe signs of clinical disease, such as severe weight loss, ruffled fur, and a hunched posture, and they succumbed to the infection or were euthanized by 6 dpi (Figure 1). The average time until death for each group decreased from 6 dpi in the low-dose challenged groups to 4 dpi in the high-dose challenged groups (Figure 1A). No significant differences were observed in the survival and weight curves depending on the route of infection (i.e., i.d., i.p., i.m., and s.c.) (Figures 1A,B). The MLD₅₀ for SFTSV SD4 in IFNAR^-/- mice inoculated via s.c. was determined as 8.7 × 10^-2 TCID₅₀ (data not shown). The IFNAR^-/- mice euthanized at the endpoint of the fatal illness were subjected to virological, hematological, and histopathological examinations in order to investigate the pathogenesis of SFTS in the model. Whole blood samples from eight mice that reached the humane endpoint [i.e., three mice from the i.p.-low (mice inoculated with a low dose of SFTSV via i.p.) group, two mice from the s.c.-low group, and one mouse each from the i.d., s.c.-high, and i.m.-low groups] were used in virus titration and those from six out of the eight mice were used in hematological examination. Regardless of the route employed, the infected mice exhibited severe viremia and lymphocytopenia (Figures 1C,D). The platelet counts (Figure 1E) for the infected mice were significantly lower than those of the control animals, and the mean platelet volumes (Figure 1F) of the infected mice were significantly higher, thereby suggesting the occurrence of platelet destruction and the activation of platelet production.

Loss of splenic white pulp and diffuse reticuloendothelial hyperplasia of the red pulp were evident in the spleens of infected mice (Figure 2A). Destruction of lymphoid follicles was observed in the cortex of the cervical lymph nodes as well. The SFTSV-infected mice developed histiocytic and necrotizing lymphadenitis lesions with pyknosis and karyorrhexis of lymphocytes in the spleen and cervical lymph nodes at the terminal stage of infection (Figure 2A, magnified figures). The subcapsular and medullary sinuses frequently contained moderate amounts of edema and fibrin with thrombosis of multiple vessels within the nodes, and histiocytic proliferation was observed in both the medulla and cortex of the affected lymph nodes. In the bone marrow, there was a paucity of erythroid precursor cells and an increase in the myeloid to erythroid ratio (Figure 2B). Minimal to moderate necrosis of the bone marrow was collocated with edema and fibrin (Figure 2B, magnified figure). B220-positive B cells in the spleen and lymph nodes of the infected mice were apparently decreased in number and distributed in the disrupted structure of each tissue (Figure 2C). Furthermore, increased numbers of IbaI-positive cells with enlarged cytoplasm, which might have been Kupfer cells, were observed in the livers of infected mice compared with those in the uninfected mice (Figure 2D).

Severe fever with thrombocytopenia syndrome virus antigen-positive cells were found in the spleen, lymph nodes, liver, and kidney (Figure 3), whereas they were not observed in the spinal cord, gastrointestinal tract, brain, and heart (data not shown). The antigen-positive cells were most abundant in the spleen among the tissues examined. Several antigen-positive cells were morphologically classified into three different cell types (i.e., monocytes, lymphocytes, and reticular cells, magnified figures in Figure 3), which were distributed diffusely in the spleen and lymph nodes. However, the necrotic cells in the lesions were rarely positive for the SFTSV antigen. Monocyte-like antigen-positive cells were frequently found surrounding the necrotic lesions in the spleen and lymph nodes. Monocyte-like antigen-positive cells were also observed in the liver but without obvious pathological changes, although the prevalence of the infected cells was clearly lower than that in the spleen and lymph nodes. The interstitial cells of the renal cortex in the kidney were positive for SFTSV antigen. The infected cells in the kidney were distributed sporadically throughout the tissue and they accumulated in the renal capsules and proximal tubules.

In order to identify the cells in which the virus replicates, double immunohistochemical staining of the host cell marker(s) and virus antigens was performed using tissues from the spleen, cervical lymph nodes, liver, and kidney (Figure 4). In the spleen, the majority of the SFTSV-infected cells overlapped with the IbaI-positive macrophages or gp36-positive reticular cells and none or a few overlapped with the CD3e- or Pax5-positive T or immature B lymphocytes (Figure 4A). Phagocytosis of the SFTSV-infected lymphocytes by IbaI-positive macrophages was detected (Figure 4A, arrow). IbaI-positive macrophages infected with SFTSV were dispersed throughout the organ. However, while the SFTSV/IbaI double positive cells were major among the SFTSV-infected cells, most of the macrophages in the spleen were not infected with the virus. A limited number of Pax5-positive B cells were found in the same lesions where extensive necrosis of B220-positive B cells (which may have included Pax5-positive cells) was observed in the spleen. Similar overlapping of the SFTSV antigens with IbaI, Pax5, and gp36 was also found in the lymph nodes (Figure 4B). There was a high level of overlapping for IbaI and SFTSV antigens in the liver (Figure 4C) and kidney (Figure 4D). A few interstitial gp36-positive reticular cells in the kidney were infected with SFTSV. Our results demonstrate that macrophages, immature B cells, and reticular cells were infected with SFTSV in the terminal stage of the IFNAR^-/- mouse model.